Compositions and methods for treating, preventing or reversing age associated inflammation and disorders

ABSTRACT

Disclosed is a method for preventing, delaying or reversing age-associated inflammation, by administering to a patient in need thereof a therapeutically effective amount of at least one reverse transcriptase inhibitor (RTI).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of InternationalApplication No. PCT/US/2020/015043 filed Jan. 24, 2020, which claimspriority from U.S. Provisional Patent Application No. 62/797,109 filedJan. 25, 2019, and U.S. Provisional Patent Application No. 62/907,251filed Sep. 27, 2019, the entire contents of which are herebyincorporated by reference herein.

FIELD OF THE INVENTION

The invention is in the field of medicinal chemistry. In particular, theinvention relates to a method for treating, preventing and/or reversinga pathophysiological L1-associated process, e.g., age-associatedinflammation, by administering a reverse transcriptase inhibitor (RTI)to a patient in need thereof. The pathophysiological L1-associatedprocess may be in a patient having Alzheimer's disease, amyotrophiclateral sclerosis (ALS), Parkinson's disease, Huntington's disease,vision loss, hearing loss, peripheral degenerative diseases, orcardiovascular dysfunction, frontotemporal dementia (FTD), multiplesclerosis (MS), Aicardi Goutiere's syndrome, progressive supra nuclearpalsy (PSP), osteoarthritis, skin aging, atherosclerosis,chemotherapy-induced adverse effects, hematopoietic stem cell function,osteoporosis, physical function, Rett Syndrome, schizophrenia, autismspectrum disorder (ADS) and/or pulmonary fibrosis, or in a patient inneed of wound healing or tissue regeneration.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was developed with the following funding: Glenn/AFARPostdoctoral Fellowship, NIH P20 GM119943 COBRE pilot award; NIH F31AG043189; NIH T32 AG041688; NIH F31 AG050365; Biotechnology and SportMedicine Fellowships, School of Pharmacy, University of Bologna,Bologna, Italy; NIH R37 AG016667, R01 AG024353, P01 AG051449, Glenn-AFARBreakthroughs in Gerontology Award; NIH R01 AG050582, P20 GM109035; NIHR37 AG016694, P01 AG051449.

BACKGROUND OF THE INVENTION

The number of people living to be 60 years or older is increasingworldwide. Between 2012 and 2050, the proportion of the number of peopleaged 60 years and over is expected to increase from 809 million to 2billion (or 11% to 22% of the population).¹ Among the leading causes ofdeath in the elderly are several chronic conditions including heartdisease, cancer, diabetes, Alzheimer's disease, and infection.Importantly, many of these age-related diseases and aging itself areclosely associated with low-level chronic inflammation.^(2,3,4) Systemicchronic inflammation can accelerate aging.⁵ Indeed, many Inflammatorymarkers are significant predictors of mortality in older humans.⁶

Despite this common link between aging, inflammation, and chronicdiseases, limited progress has been made to understand the mechanismsthat control age-related inflammation, and the causal relationship ofthese regulators to chronic degenerative diseases is not completelyunderstood. A better understanding of the role of these regulators inage-related inflammation should lead to new strategies for extending thehealth of the older population.

As such, there is a need in the art for better treatment and preventionof age-related inflammation and age-related disorders.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a better understanding of the mechanismsunderlying age-related inflammation and its role in the aging, and otherpathophysiological processes related to LINE-1 (L1) retrotransposons, aswell as compositions and methods for treating, preventing and/orreducing age-associated inflammation and other disorders.

Retrotransposable elements (RTEs) are deleterious at multiple levels,and failure of host surveillance systems can thus have negativeconsequences. However, the contribution of RTE activity to aging andage-associated diseases was not known. The present invention is based onseveral empirical observations including that, during cellularsenescence, LINE-1 (L1) elements become transcriptionally upregulatedand activate a type-I interferon (IFN-I) response. The IFN-I response isa novel phenotype of late senescence and contributes to the maintenanceof the senescence associated secretory phenotype (SASP). The IFN-Iresponse is triggered by cytoplasmic L1 cDNA and is antagonized byreverse transcriptase inhibitors (RTIs) that inhibit the L1 reversetranscriptase (RT). Treatment of aged mice with the RTI lamivudinedownregulated IFN-I activation and age-associated inflammation inseveral tissues. As such, RTE activation is an important component ofsterile inflammation that is a hallmark of aging, and L1 RT is arelevant target for the treatment of age-associated disorders.

The present invention provides a method for treating, preventing and/orreversing a pathophysiological L1-associated process such asage-associated inflammation in a patient in need thereof byadministering a therapeutically-effective amount of at least one reversetranscriptase inhibitor (RTI) to the patient.

In a comparative assessment of several RTI drugs in a dose-responseassay of the inhibition of L1 activity, three RTI drugs, islatravir,censavudine and elvucitabine, displayed unexpected ability to inhibitmouse and human L1 activity. In particular, islatravir was asurprisingly potent inhibitor of human L1. The present invention furtherprovides a method for treating, preventing and/or reversingpathophysiological L1-associated processes such as age-associatedinflammation in a patient in need thereof by administering atherapeutically-effective amount of islatravir and/or censavudine and/orelvucitabine to the patient.

Without wishing to be bound by any particular theory, thepathophysiological L1-associated process, e.g., age-associatedinflammation, is associated with an upregulation of L1, an accumulationof cytoplasmic L1 cDNA, an activation of an IFN-I response, and/or areinforcement of a SASP pro-inflammatory state. The RTI drug isadministered in an amount sufficient to prevent or reverse at least oneof the upregulation of L1, the accumulation of cytoplasmic L1 cDNA, theactivation of the IFN-I response, and/or the SASP pro-inflammatorystate.

The pathophysiological L1-associated processes, that can be prevented,treated, or reversed with the methods of the present invention is in apatient having a disease or disorder including, but not limited to:Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson'sdisease, Huntington's disease, vision loss, hearing loss, peripheraldegenerative diseases, or cardiovascular dysfunction, frontotemporaldementia (FTD), multiple sclerosis (MS), Aicardi Goutiere's syndrome,progressive supra nuclear palsy (PSP), osteoarthritis, skin aging,atherosclerosis, chemotherapy-induced adverse effects, hematopoieticstem cell function, osteoporosis, physical function, Rett Syndrome,schizophrenia, autism spectrum disorder (ADS) and/or pulmonary fibrosis,or in a patient in need of wound healing or tissue regeneration. In oneembodiment, the age-associated inflammation is in a patient havingAlzheimer's disease. In an alternate embodiment, the age-associatedinflammation is in a patient having ALS.

Also provided is a method for delaying or reversing the progression ofthe underlying pathology of disease disorder caused by age-associatedinflammation, comprising administering to a patient in need thereof atherapeutically effective amount of at least one RTI. In someembodiments, the patient has Alzheimer's disease or ALS and experiencesa decrease in one or more symptoms of Alzheimer's disease or ALScompared to before the first administration of the RTI to the patient.In some embodiments, the one or more symptoms of Alzheimer's diseasecomprise memory loss, misplacing items, forgetting the names of placesor objects, repeating questions, being less flexible, confusion,disorientation, obsessive behavior, compulsive behavior, delusions,aphasia, disturbed sleep, mood swings, depression, anxiety, frustration,agitation, difficulty in performing spatial tasks, agnosia, difficultywith ambulation, weight loss, loss of speech, loss of short term memoryor loss of long term memory.

In some embodiments, the decrease in the one or more symptoms ofAlzheimer's disease is evaluated according to the DSM-5.⁷ In someembodiments, the decrease of symptoms is determined using the cognitivesubscale of the Alzheimer's Disease Assessment Scale (ADAS-cog). In someembodiments, the decrease of symptoms is determined using theClinician's Interview-Based Impression of Change (CIBIC-plus). In someembodiments, the decrease of symptoms is determined using the Activitiesof Daily Living (ADL) scale. In some embodiments, the decrease ofsymptoms is for 1-36 months.

In some embodiments, any change in the underlying pathology isidentified by detection of a biomarker before and after the RTIadministration. In some embodiments, the biomarker is β-amyloid or Tauprotein. In some embodiments, the biomarker is detected by PET imaging.In some embodiments, the underlying pathology is identified bymeasurement of β-amyloid or Tau protein in the cerebrospinal fluid. Insome embodiments, the underlying pathology is identified by measurementof brain volume before and after the RTI administration. In someembodiments, the underlying pathology is reversed or delayed for at 1-36months.

In some embodiments, the at least one RTI is a nucleoside reversetranscriptase inhibitor (NRTI). In some embodiments, the at least oneNRTI is selected from: abacavir (ZIAGEN™), abacavir/lamivudine(Epzicom), abacavir/lamivudine/zidovudine (TRIZIVIR™), adefovir,alovudine, amdoxovir, apricitabine, ATRIPLA®, BARACLUDE®, BIKTARVY®,censavudine, COVIRACIL™, DAPD/DXG, D-D4FC, dexelvucitabine, didanosine(VIDEX™), didanosine extended-release (Videx EC), dOTC, EFdA(islatravir), emtricitabine (EMTRIVA™), emtricitabine/tenofoviralafenamide (DESCOVY®), emtricitabine/tenofovir disoproxil fumarate(TRUVADA®), elvucitabine, fosalvudine, lamivudine/zidovudine(COMBIVIR™), EVIPLERA™, GENVOYA®, HIVID™, KIVEXA™, lamivudine (EPIVIR™),LODENOSINE™, ODEFSEY®, PREVEON®, racivir, stampidine, stavudine(ZERIT™), STRIBILD®, TENOFOVIR™, tenofovir disoproxil fumarate(VIREAD™), TRIUMEQ®, Trizivir, VEMLIDY®, Telbivudine and/orzidovudine(RETROVIR™). In some embodiments, the at least one NRTI is censavudine.In some embodiments, the at least one NRTI is elvucitabine. In someembodiments, the at least one NRTI is EFdA (islatravir).

In some embodiments, the at least one RTI is a non-nucleoside reversetranscriptase inhibitor (NNRTI). In some embodiments, the at least oneNNRTI is selected from: Delavirdine (DLV), Efavirenz (EFV), Etravirine(TMC125), Nevirapine (NVP), and/or Rilpivirine.

In some embodiments, the patient has Alzheimer's disease and the methodfurther comprises administering at least one second therapeutic agentuseful for the treatment of the symptoms of Alzheimer's disease. In someembodiments, the at least one second therapeutic agent is selected from:donepezil, galantamine, memantine, and/or rivastigmine. In someembodiments, the at least one second therapeutic agent is an antibodythat binds to β-amyloid or Tau protein. In some embodiments, theantibody binds to β-amyloid and is bapineuzumab. In some embodiments,the antibody binds to Tau protein and is ABBV-8E12. In some embodiments,the at least one second therapeutic agent is a vaccine against β-amyloidor Tau protein. In some embodiments, the at least one second therapeuticagent is an agent that reduces or alters the brain content of β-amyloidor Tau. In some embodiments, the second therapeutic agent reduces oralters the brain content of β-amyloid and is a β-secretase 1 (BACE)inhibitor. In some embodiments, the BACE inhibitor is selected from:CTS-21166, lanabecestat (AZD3293), LY2886721, and verubecestat(MK-8931). In some embodiments, the second agent reduces or alters thebrain content of Tau and is nicotinamide, or MPT0G211. In someembodiments, the at least one second therapeutic agent is anInterferon-gamma antibody. In some embodiments, the at least one secondtherapeutic agent is a JAK/STAT pathway inhibitor.

In some embodiments, the patient has ALS and the method furthercomprises administering at least one second therapeutic agent useful forthe treatment of the symptoms of ALS. In some embodiments, the at leastone second agent useful for the treatment of ALS is edaravone and/orriluzole. In other embodiments, the at least one second agent is anintegrase inhibitor. In some embodiments, the integrase inhibitor isselected from: aurintricarboxylic acid, derivatives ofaurintricarboxylic acid, BMS-538158, caffeic acid phenethyl ester,derivatives of caffeic acid phenethyl ester, curcumin, derivatives ofcurcumin, chicoric acid, derivatives of chicoric acid,3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic acid,GSK364735C, L-870812, and L-25 870810, MK-0518, quercetin, derivativesof quercetin, raltegravir, S-1360, tyrphostin, derivatives oftyrphostin, and/or zintevir (AR-177).

In some embodiments, the patient is evaluated for one or more symptomsor disease pathology for 1-36 months after the first administration tothe patient of the RTI.

In some embodiments, the RTI inhibits L1 reverse transcriptase activityin a cell of the patient.

Also provided is a method for preventing the onset of Alzheimer'sdisease in a patient suspected of having mild cognitive impairment orpreclinical Alzheimer's disease, comprising administering atherapeutically effective amount of at least one RTI to a patient inneed thereof.

Other implementations are also described and recited herein.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustration, certain embodiments of the presentinvention are shown in the drawings described below. Like numerals inthe drawings indicate like elements throughout. It should be understood,however, that the invention is not limited to the precise arrangements,dimensions, and instruments shown. In the drawings:

FIGS. 1A-1E show the activation of L1, IFN-I and SASP in senescentcells. Gene expression was assessed by RT-qPCR. Poly(A)-purified RNA wasused in all L1 assays. FIG. 1A is a line graph showing the time courseof L1 activation. P values were calculated relative to EP, early passagecontrol. FIG. 1B is a schematic of L1 RT-PCR strategy. For primerspecificity, see FIGS. 6F-H; for primer design, see Methods. Primers foramplicon F were used in (a) and (e). FIG. 1C is three bar graphs showingstrand-specific L1 transcription was assessed using amplicons A-F.Transcription from the 5′UTR antisense promoter was also detected. SEN(L), late senescence (16 weeks). FIG. 1D is a bar graph showinginduction of IFN-α and IFN-β1 mRNA levels. FIG. 1E is three imagesshowing the temporal induction of genes associated with DNA damage (p21,also known as CDKN1A), SASP (IL-1β, CCL2, IL-6, MMP3), and the IFN-Iresponse (IRF7, IFN-α, IFN-β1, OAS1). Row clustering was calculated as1-Pearson correlation. RS, replicative senescence; OIS, oncogene inducedsenescence (elicited by Ha-RAS infection); SIPS, stress inducedpremature senescence (gamma irradiation). Controls: EP, early passage;EV, empty vector infected; CTR, non-irradiated. (FIGS. 1A, 1C-E), n=3independent biological samples, repeated in two independent experiments.(FIGS. 1A, 1C, 1D) Data are mean±s.d. *P≤0.05, **P≤0.01, unpairedtwo-sided t-tests.

FIGS. 2A-H show the regulation of L1 activation and IFN-I induction.FIG. 2A is two bar graphs showing expression and ChIP of RB1 and FIG. 2Bis two bar graphs showing FOXA1 expression was measured by RT-qPCR andimmunoblotting (left panels). Binding to L1 elements was assessed withChIP-qPCR (right panels). For primer specificity, see FIG. 6B RB1:5′UTR, ORF1 and ORF2, primers for amplicons A, E and F, respectively.FOXA1: primers for amplicons A-E. qPCR was normalized to inputchromatin. SEN (E), early senescence (8 weeks). For gel source, data seeFIG. 16 . FIG. 2C is a bar graph, and FIGS. 2D-F are each two bar graphsshowing RB1, FOXA1 or TREX1 were overexpressed (OE) or ablated withshRNAs and the effects on expression of L1, IFN-α and IFN-β1 weredetermined by RT-qPCR of poly(A)-purified RNA. In all cases, lentiviralvectors were used to deliver the interventions directly into senescentcells at 12 weeks (point D, FIG. 6A), and cells were harvested foranalysis four weeks later (point E, 16 weeks). Controls were uninfectedsenescent cells harvested at the same time (point E, 16 weeks). Twodistinct shRNAs (a, b) were used for each gene. Primers for amplicon Fwere used for L1. FIG. 2F is two bar graphs showing RB1 wasoverexpressed as above and its binding to the 5′UTR was assessed byChIP-qPCR (amplicon A). FIG. 2G is two bar graphs showing activation ofL1, IFN-α and IFN-β1 expression after the triple (3×) intervention usingshRB1 (a), shTREX1 (a) and FOXA1-OE in early passage cells. Lentiviralinfections were performed sequentially with drug selections at each step(shRB1, puromycin→shTREX1, hygromycin→FOXA1-OE, blasticidin). FIG. 2H istwo line graphs showing expression of IFN-I pathway genes was determinedwith the RT2 Profiler PCR array (Qiagen). Normalized mean expression isshown for all 84 genes in the array. Red symbols: significantlyupregulated genes. Dashed lines demarcate the ±2-fold range. (FIGS. 2A,2B, 2H), n=3 independent biological samples, repeated in 2 independentexperiments. (FIGS. 2C-G), n=3 independent experiments. (FIGS. 2A-G)Data are mean±s.d. *P≤0.05, **P≤0.01, unpaired two-sided t-tests.

FIGS. 3A-F show that the ablation of L1 relieves IFN-I activation andblunts the SASP response. FIG. 3A is twelve images showing cells wereexamined by immunofluorescence (IF) microscopy using antibodies tosingle-stranded DNA (ssDNA) or L1 ORF1 protein. Note the bright ssDNApuncta in senescent cells that colocalized with prominent puncta ofORF1. The experiment was independently repeated 3 times with similarresults. Scale bar=10 μm. FIG. 3B is three bar graphs showing senescentcells were treated with L1 shRNAs (using lentiviral vectors as describedin FIGS. 2C, 2E, 2F) or with 3TC (7.5 μM) between 12 and 16 weeks ofsenescence. Effects on the IFN-I response were determined by RT-qPCR,ELISA or immunoblotting. For gel source data, see FIG. 16 . FIG. 3C is abar graph showing cells were labeled with BrdU for 2 weeks (with orwithout 7.5 μM 3TC), labeled DNA was immunoprecipitated, and its L1sequence content was quantified using a TaqMan multiplex qPCR assay 16(FIG. 1B, amplicon F). EP (qui), early passage quiescent cells. FIG. 3Dis two bar graphs showing Left panel, RS cells: IFNAR1 and IFNAR2 geneswere mutagenized using the CRISPR/Cas9 system delivered with lentivirusvectors directly into senescent cells. As with shRNA interventions,cells were infected at 12 weeks and harvested at 16 weeks of senescence(see FIGS. 1D-F, see Methods). Right panel, SIPS cells: CRISPR/Cas9intervention was performed in early passage cells and a validated clonewas irradiated to induce SIPS. FIG. 3E is two bar graphs showing OIS andSIPS were induced as in FIG. 1D and cells were harvested 20 days (OIS)or 30 days (SIPS) later. 3TC (7.5 μM) was present throughout. IFN-I geneexpression (IFN-α, IRF7, OAS1) was measured by RT-qPCR. FIG. 3F is fourbar graphs showing cells were serially passaged into replicativesenescence (RS) with 3TC (10 μM) present throughout, and the temporalinduction of SASP response genes (IL-1β, CCL2, IL-6, MMP3) was assessed.(FIGS. 3B-D, F), n=3 independent experiments. (FIG. 3E) n=3 independentbiological samples, repeated in 2 independent experiments. (FIGS. 3B-F)Data are mean±s.d. *P≤0.05, **P≤0.01. (FIGS. 3B, D-F) unpaired two-sidedt-tests, (c) 1-way ANOVA with Tukey's multiple comparisons test.

FIGS. 4A-F show that L1s are activated with age in murine tissues andthe IFN-I proinflammatory response is relieved by RTI treatment. FIG. 4Ais six images and a table showing presence of L1 Orf1 protein in tissueswas examined by IF microscopy. Quantification of ORF1 expressing cellsis shown in the right panel; three animals and at least 200 cells peranimal were scored for each condition. Scale bar=4 μm. FIG. 4B is siximages and a table showing activation of L1 in senescent cells wasexamined by co-staining for SA-β-Gal activity and Orf1 protein by IF(male liver, 5 and 26 months). Scale bar=4 μm. The experiment wasrepeated three times independently with similar results. FIG. 4C isthree box plots showing mice were administered 3TC (2 mg/ml) in drinkingwater at the indicated ages for two weeks and sacrificed aftertreatment. Expression of p16, an IFN-I response gene (IFN-α), and amarker of a proinflammatory state (11-6) were assessed by RT-qPCR. SeeFIG. 14 for additional tissues and genes. The box plots show the rangeof the data (whiskers), 25th and 75 percentiles (box), means (dashedline), and medians (solid line). Each point represents one animal. 5months, n=8; 26 months, n=12; 29 months, n=6. FIG. 4D is four box plotsshowing six-month-old were non-lethally irradiated and expression of L1,p16 and representative IFN-I response genes (Ifn-α, Oas1) were assessedby RT-qPCR at the indicated times post-irradiation. Graphicalpresentation is as in (c); non-irradiated, n=3 animals at 3 months, n=5animals at 6 months; irradiated, n=4 animals at 3 months, n=5 animals at6 months. FIG. 4E is four box plots and two images showing macrophageinfiltration into white adipose tissue and kidney was scored as F4/80positive cells (% of total nuclei). n=5 animals group (adipose); n=8(kidney). Skeletal muscle fiber diameter was measured (see Methods fordetails) and plotted as an aggregate box plot. n=5 animals per group,500 fibers total. Glomerulosclerosis was scored in periodic acid-Shiff(PAS)-stained sections (see Methods for details) as the sum of allglomeruli with a score of 3 or 4 divided by the total. n=7 animals pergroup, 40 glomeruli per animal. Graphical presentation is as in (c). 3TCtreatment was 2 weeks for white adipose and 6 months (20-26 months) forother tissues. Dashed circle demarcates a single glomerulus. Scalebar=50 μm. FIG. 4F is a schematic showing breakdown of L1 surveillancemechanisms leads to chronic activation of the IFN-I response. ISD:interferon-stimulatory DNA pathway. *P≤0.05, **P≤0.01, unpairedtwo-sided t-tests (FIGS. 4A, 4D, 4E) or 1-way ANOVA with Tukey'smultiple comparisons test (FIGS. 4C, 4E white adipose).

FIG. 5 is a flow chart outlining the molecular pathway of cellularsenescence leading to age-associated, “sterile” inflammation.

FIGS. 6A-K show the establishment of senescent cultures and analysis ofL1 and IFN-I activation. FIG. 6A is a line graph showing the passagingregimen to obtain long-term replicatively senescent cells (details inMethods). Point A was designated as zero for time in senescence.Confirmation of the senescent status of cultures. A representativeexperiment is shown; other experiments were monitored in the same mannerand generated data that met these benchmarks. EP, early passage control;SEN (E), early senescence (8 weeks); SEN (L), late senescence (16 weeksFIG. 6B is three bar graphs showing cells were labeled with BrdU for 6hours. BrdU incorporation⁸ and senescence-associated β-galactosidase(SA-β-Gal) activity⁹ were determined as indicated. DNA damage foci werevisualized using γ-H2AX antibodies and immunofluorescence microscopy(IF).¹⁰ FIG. 6C is two bar graphs showing expression of p21 (CDKN1A) andp16 (CDKN2A) proteins was determined by immunoblotting. GAPDH was theloading control. For gel source data, see FIG. 16 . FIG. 6D is a bargraph showing expression of genes characteristic of the SASP wasdetermined by RT-qPCR. FIG. 6E is two bar graphs showing L1 activationduring senescence of IMR-90 and WI-38 strains of fibroblasts wasassessed by RT-qPCR using poly(A) purified RNA and primers for ampliconF (FIG. 1B). FIG. 6F is a graph showing long-range RT-PCR was performedwith primers A-forward and C-reverse (amplicon G) and primers A-forwardand D-reverse (amplicon H) (FIG. 1B, Table 1) and the cDNAs were clonedand sequenced. Several attempts using the same protocol on early passageproliferating cells did not yield any L1 clones. Sequences were mappedto the unmasked reference genome demanding 100% identify. 658 clonescould be thus mapped, 51 additional clones contained at least 1 mismatchand thus likely represent elements that are polymorphic in the cellline, and 58 were cloning artifacts. Among the 658 mappable clones 224unique elements were represented (Table 3). Intact elements are thesubset of full-length elements annotated with no ORF inactivatingmutations. Size of the features corresponds to the number of times theelement was represented among the 658 clones. FIG. 6G is a table showingthe summary of long-range PCR data presented in FIG. 6F and Table 3.FIG. 6H is a table showing apparent genomic copy numbers of elementsdetected with our amplicons (see FIG. 1 b for locations of amplicons andMethods for primer design strategy). Predicted: in silico PCR (seeMethods for details). Observed: qPCR was performed on 1 ng of genomicDNA and normalized to a known single copy locus. FIG. 6I is a two bargraphs showing activation of IFN-α and IFN-β1 genes during senescence ofWI-38 and IMR-90 cells was determined by RT-qPCR. FIG. 6J is two bargraphs showing confirmation of the senescent status of cells in OIS (20days, FIG. 6E) and SIPS (30 days, FIG. 6E) by SA-β-Gal activity. EV,empty vector control; CTR, non-irradiated cells. FIG. 6K is three bargraphs showing confirmation of full length L1 mRNA expression in allforms of senescence using RT-qPCR with primers for amplicons A and F onpoly(A)-purified RNA. Late onset activation is shown by comparing days 9and 20 for OIS and days 12 and 30 for SIPS. (FIGS. 6B-E, 6I-K), n=3independent biological samples, repeated in 2 independent experiments.Data are mean±s.d. *P≤0.05, **P≤0.01, unpaired two-sided t-tests.

FIGS. 7A-B show the mapping of transcriptional start sites in L1elements activated during cellular senescence. 5′RACE was performed withprimers C and D (FIG. 1A, Table 1) on late senescent cells (16 weeks,point D in FIG. 6A), the products were cloned, and individual cloneswere Sanger sequenced (see Methods for details). FIG. 7A is a series ofimages showing a multiple sequence alignment of the 50 mappable clonesagainst the L1HS consensus was generated with MAFFT software. The L1HSconsensus is shown on top. Blue color shading of the aligned clonesshows their degree of identity with the consensus. The green verticalline marks the start (position 1) of the L1HS consensus. Red verticallines mark short gaps (1-4 nucleotides) opened in the L1HS consensus byindividual clones. The consensus of the 50 clones is shown at the bottomand was generated with Jalview. The initiation of L1 transcription isknown to be imprecise, with the majority of start sites occurring +/−50bp of the consensus start site, and a subset as far down as +180 bp.¹¹FIG. 78 is a table showing the summary of the mapping data andclassification of clones to families of L1 elements. The relative startsites were calculated relative to the L1HS consensus start site.RepEnrich software¹² was used to assign the clones to L1 families.

FIGS. 8A-G show the evolution of transcriptomic changes duringprogression of cellular senescence. RNA-seq was performed on earlyproliferating LF1 cells (EP) and cultures at 8 weeks (SEN-E) and 16weeks (SEN-L) in senescence (points C and D, respectively, in ExtendedData FIG. 6A). Data were analyzed using a 3-way comparison: EP versusSEN-E, EP versus SEN-L and SEN-E versus SEN-L (see Methods for details).FIG. 8A is a series of six images showing area-proportional generalizedVenn diagrams depicting the intersections of the three comparisons forthe following datasets. i-ii, significantly upregulated anddownregulated genes (row 2× in panel b). iii-iv, significant KEGGpathways identified by GSEA. Note the considerable evolution thetranscriptome in late senescence, exemplified by large changes(especially upregulated) in differentially expressed genes as well aspathways. v-vi, significantly changing genes in the IFN-I and SASP genesets (see Table 4 for annotation of gene sets). Note that the majorityof changes in SASP genes occur early, whereas a large component of IFN-1changes is specific for late senescence. FIG. 8B is a table showing thesummary of significantly changing genes using a fixed FDR (<0.05) andvariable fold-change cutoffs (2×, 1.75× and 1.5×). FIG. 8C is an imageshowing GSEA analysis of KEGG pathways. Heatmap representation showssignificantly upregulated pathways in red (also see panel e) anddownregulated pathways in blue. Non-significant comparisons are shown inblack; vertical annotations refer to Venn diagrams in (a, iii-iv). Notethat the SASP gene set is upregulated early whereas the IFN-1 gene setis upregulated late. FIG. 8D is an image showing heatmaps ofsignificantly changing genes in the IFN-1 and SASP gene sets. Verticalannotations refer to Venn diagrams in (a, v-vi). FIG. 8E is three tablesshowing the list of significantly upregulated KEGG pathways identifiedusing GSEA (see Table 5 for a list of all pathways). NES, normalizedenrichment scores. IFN-1 and SASP gene sets are highlighted in yellow.Note the significant upregulation of IFN-1 between early and latesenescence. Red type identifies KEGG pathways indicative of cytosolicDNA sensing and a type I interferon response at late times. FIG. 8F isthree images and FIG. 8G is three images showing GSEA profiles of theIFN-I and SASP genesets for all comparisons; FDR is highlighted inyellow. Note that the upregulation of IFN-I is significant for EP_SEN-Land SEN-E_SEN-L but not for EP_SEN-E, and that the upregulation of SASPis significant for EP_SEN-E and EP_SEN-L but not SEN-E_SEN-L. n=3independent biological samples. Differential expression data wereanalyzed for significance using the GSEA GenePattern interface and theoutputs were corrected for multiple comparisons by adjusting the nominalp values using the Benjamini-Hochberg method (see Methods for details).

FIGS. 9A-K show the characterization of L1 effectors and the IFN-Iresponse. FIG. 9A is a bar graph showing expression of TREX1 wasdetermined by RT-qPCR and immunoblotting. For gel source data, see FIG.16 . FIG. 9B is a bar graph showing expression of RB family genes werecompared by RT-qPCR. Primer pairs for all genes were verified to be ofequivalent efficiency. FIG. 9C is two bar graphs showing enrichment ofH3K9me3 and H3K27me3 on L1 elements was examined by ChIP-qPCR (PCRprimers illustrated in FIG. 1B were used: 5′UTR, amplicon A; ORF1,amplicon E; ORF2, amplicon F). FIG. 9D is an image showing ChIP-seq datafrom ENCODE were investigated for transcription factors that bind to theL1 consensus sequence. The log fold change enrichment relative to inputcontrols is shown for the indicated cell-lines. The binding of YY1 tothe L1 promoter has been documented¹³ and was used as a positivecontrol. CEBPB was used as a negative control. A schematic illustratingL1 coordinates and relevant features is shown above. Amplicons A-E arethe same as shown in FIG. 1B. FIG. 9E is two bar graphs showingtranscriptional activity of the intact L1 5′UTR or a UTR lacking theFOXA1 binding site (UTR-A) was determined using sense and antisensereporters cotransfected into early passage LF1 cells either with a FOXA1expression plasmid or empty vector (EV). FIG. 9F is a bar graph showingFOXA1 was knocked down in senescent cells with shFOXA1 (a) (see alsoFIG. 2E and FIG. 10A) and binding to the L1 5′UTR (amplicon B) wasdetermined by ChIP-qPCR. FIG. 9G is three bar graphs showing knockdownof RB1, TREX1 and ectopic expression of FOXA1 were performed in earlypassage cells in all single (1×), double (2×) and triple (3×)combinations and assessed by RT-qPCR using poly(A)-purified RNA foractivation of L1, IFN-α and IFN-β1 expression (primers for amplicon F).Three controls are shown: cells infected with irrelevant shRNA (shGFP),expression construct (LacZ), or uninfected early passage cells (EP).FIG. 9H is two bar graphs showing L1 5′UTR occupancy of RB1 and FOXA1 in3× cells was determined by ChIP-qPCR performed as in FIGS. 2A, 2B.Primers for amplicons A and B were used for RBI and FOXA1, respectively.For comparison, single interventions in early passage cells with shRB1(a) or FOXA1 cDNA expression (EP FOXA1-OE) are also shown. FIG. 9I is abar graph showing confirmation of full length L1 mRNA expression in 3×cells using RT-qPCR with primers for amplicons A and F onpoly(A)-purified RNA. CTR, cells infected with irrelevant shRNA (shGFP).FIG. 9J is an image of a heat map representation showing all biologicalreplicates for the 67 genes significantly changing expression in SENand/or 3× cells (FIG. 2H, Table 6). Column clustering was calculated as1-Pearson correlation. Rows have been grouped into functional subsets ofthe IFN-I response. FIG. 9K is a Venn diagram showing the overlapbetween the 67 significantly changing genes. (FIGS. 9A-F, 9H) n=3independent biological samples, repeated in two independent experiments.(FIGS. 9G, 9I), n=3 independent experiments. (FIGS. 9A-1 ) Data aremean±s.d. *P≤0.05, **P≤0.01, unpaired two-sided t-tests.

FIGS. 10A-M show the efficacy of genetic and pharmacologicalinterventions. FIG. 10A is three bar graphs showing knockdowns with twodistinct shRNAs (a, b) or FIG. 10B is three bar graphs showing ectopiccDNA expression were performed in senescent cells as described in FIGS.2D, 2E, 2G (also see Methods). The effectiveness of these manipulationson their targets was assessed by RT-qPCR and immunoblotting. For gelsource data see FIG. 16 . FIG. 10C is three bar graphs showing RB1,TREX1 and FOXA1 mRNA and protein expression after the triple (3×)intervention (FIG. 2F). FIG. 10D is a line graph showing the effect of3TC treatment on the relative abundance of L1HS sequences in senescentcells was determined by multiplex TaqMan qPCR on total DNA (primer set6, Table 1). SEN entry, 0 weeks in senescence (FIG. 1A; point A in FIG.6A). 3TC was administered continuously from SEN entry until harvest 16weeks later. FIG. 10E is a line graph showing the dual luciferase L1reporter system¹⁴ was used to determine the effect of 3TC dosing onretrotransposition. L1 reporters were introduced into early passagecells using lentivirus vectors (see Methods for details) and cells weretreated with 3TC for 4 days prior to harvest and assay. JM111, adefective reporter carrying mutations in ORF1 (absence of 3TC); L1RP, aretrotransposition competent reporter. FIG. 10F is a line graph showingthe effect of 3TC dosing on the IFN-I response. The experiment above (d)was processed by RT-qPCR to determine the expression of IFN-α andIFN-β1. FIG. 10G is two bar graphs showing knockdowns of L1 wereperformed with two distinct shRNAs (a, b) in senescent cells (as inFIGS. 2D, 2E, 2G) or 3× cells (as in FIG. 2G). The effectiveness on L1expression was assessed by RT-qPCR using poly(A)-purified RNA andprimers F. FIG. 10H is nine images and a bar graph showing cells in theexperiment in (g) were examined for levels of ORF1 protein byimmunofluorescence (IF). Image analysis was performed with CellProfilersoftware (see Methods for details). >200 cells were examined for eachcondition (a.f.u., arbitrary fluorescence units). FIG. 10I is a bargraph showing the L1 shRNA treatment in the experiment in (g) wassubstituted with 3TC treatment (10 μM) for the same period of time. FIG.10J is two bar graphs showing five different RTIs (or combinations) weretested for effects on the IFN-I response. AZT (Zidovudine, 15 μM), ABC(Abacavir, 15 μM), FTC (Emtricitabine, 10 μM), 3TC (aka Lamivudine orEpivir, 10 μM), TZV (Trizivir, a combination of 15 μM AZT, 15 μM ABC and7.5 μM 3TC). Cells were treated for 4 weeks between 12 and 16 weeks insenescence (FIG. 1A; points D and E in FIG. 1A). 3× cells (FIG. 2F) weretreated with 3TC for 48 hours after the completion of the last drugselection. IFN-α expression was determined by RT-qPCR. FIG. 10K is a bargraph showing a native L1 reporter (pLD143)¹⁵ was co-transfected withshRNA plasmid vectors into HeLa cells (see Methods for details).Retrotransposition was scored as GFP-positive cells, and shL1 knockdownswere normalized to a shLuc negative control. The absolute averageretrotransposition frequency (percentage of GFP-positive cells) was 4.1,which matches the published values for the reporter used (pLD143)53.FIG. 10L is two bar graphs showing knockdowns of cGAS and STING wereperformed in senescent or 3× cells as with the other shRNAs (FIG. 2D,2E, 2G and FIG. 10A, 10G above). FIG. 10M is 18 images showingdownregulation of interferon signaling after CRISPR-mediatedinactivation of IFNAR1 and IFNAR2 genes was verified by the absence ofIRF9 nuclear translocation and STAT2 phosphorylation in response tointerferon stimulation. Cells were infected with lentivirus vectorsexpressing Cas9 and gRNAs to both IFNAR1 and IFNAR2 (ΔIFNAR, seeMethods). After the infection cells were re-seeded on coverslips,treated with interferon for 2 hours, and examined by IF microscopy. Theexperiment was repeated 3 times with similar results. (FIGS. 10A-I, L)n=3 independent experiments. (FIG. 10K) n=3 independent biologicalsamples, repeated in 2 independent experiments. (FIGS. 10A-I) Data aremean±s.d. *P≤0.05, **P≤0.01, unpaired two-sided t-tests.

FIGS. 11A-H show the characterization of cytoplasmic DNA in senescentcells. FIG. 11A is nine images and a bar graph showing quiescent andsenescent cells were treated with BrdU as in FIG. 3A and the cellularlocalization of BrdU incorporation was visualized by IF microscopy.Proliferating cells, EP(Prol), are shown as a positive control fornuclear BrdU incorporation. The signals were quantified usingCellProfiler software (right panel, see Methods). >200 cells wereexamined for each condition (a.f.u., arbitrary fluorescence units). FIG.11B is two bar graphs showing senescent (and EP control) cells (neitherlabeled with BrdU) were fractionated into nuclear and cytoplasmicfractions, and the representation of L1 sequences in these compartments(as well as whole cells) was assessed with qPCR as in FIG. 3A (TaqManmultiplex qPCR assay 16, amplicon F, FIG. 1B). Note that the Y axisunits differ by 10-fold between the left and right panels. FIG. 11C istwelve images showing cells were examined by IF microscopy for thepresence of ORF1 protein, RNA-DNA hybrids, and single-stranded DNA(ssDNA). See Methods and Table 2 for antibodies. The RNA-DNA signal insenescent cells largely colocalized with the ORF1 signal and was lostafter RNase A treatment. The ssDNA signal also colocalized with the ORF1signal and was exposed by RNase treatment. The experiment was repeatedthree times with similar results. FIG. 11D is an image showing thepulled-down BrdU-containing DNA (FIG. 3C, panel (a) above, see Methods)was cloned and Sanger sequenced. Of the 96 total clones examined, 37mapped to L1. Red boxes represent the relative positions of these cloneson the L1 consensus sequence. FIG. 11E is a bar graph showing senescentcells labeled with BrdU (FIG. 3C, panel (a) above) wereimmunoprecipitated with anti-BrdU antibodies, and the representation ofL1 sequences in the pulled-down DNA was assessed using qPCR with primersspanning the entirety of L1 elements (FIG. 1B, 1C). FIG. 11F is a bargraph showing senescent cells were treated with L1 shRNA (usinglentiviral vectors as described in FIG. 10G) between 12 and 16 weeks ofsenescence, and expression of SASP genes was determined. FIG. 11G isthree bar graphs showing transcription throughout murine L1 elements wasassessed in a strand-specific manner using the same strategy as wasapplied to human L1 elements (FIG. 1B, 1C). The amplicons (designatedW-Z to distinguish them from the human-specific primers) correspond tothe 5′UTR (W), Orf1 (X), Orf2 (Y) and 3′UTR (Z). Also see Methods andTable 1 for primer sequences (primer sets 37, 48-50). Poly(A) RNA wasprepared from male white adipose tissue. A total of 12 animals wereassessed (3 pools of 4 animals each) in three independent experiments.FIG. 11H is a bar graph showing expression of the three currently activefamilies of murine L1 elements. Primers were designed to distinguishing5′UTR polymorphisms of the MdA, MdN and Tf families (see Methods, Table1 primer sets 51-53). RT-qPCR was performed as in (f) above(non-strand-specific). (FIGS. 11A, 11B, 11E) n=3 independent biologicalsamples, repeated in two independent experiments. (FIG. 11F), n=3independent experiments. (FIGS. 11A, 11E-H) Data are mean±s.d. *P≤0.05,**P≤0.01. (FIG. 11A) 1-way ANOVA with Tukey's multiple comparisons test,(FIGS. 11B, 11E-H) unpaired two-sided t-tests.

FIGS. 12A-G show the effects of ablating L1 activation, the cytoplasmicDNA sensing pathway, or interferon signaling on expression of the IFN-Iand SASP responses. FIG. 12A is three bar graphs showing 3× cells weretreated with L1 shRNA or with 3TC for 48 hours as described in FIGS.10G, 10I. Effects on the IFN-I response were determined by RT-qPCR,ELISA or immunoblotting. For gel source data, see FIG. 16 . FIG. 12B istwo bar graphs showing cells were serially passaged into replicativesenescence (RS) with 3TC (10 μM) present throughout as in FIG. 3F, andexpression of Cdk inhibitors p21 and p16 was assessed by RT-qPCR. FIG.12C is two bar graphs showing senescent cells were treated with shRNAsagainst cGAS or STING between 12 and 16 weeks of senescence (asdescribed in FIG. 10L), and expression of IFN-I response genes (IFN-α,IRF7, OAS1) was determined. FIG. 12D is two bar graphs showing cGAS andSTING knockdowns were performed with shRNAs in 3× cells (as in panel (c)above), and expression of IFN-I genes was examined by RT-qPCR. FIG. 12Eis a bar graph showing cGAS and STING were knocked down in senescentcells with shRNAs (as in panel (c) above) and expression of SASPresponse genes (IL-1β CCL2, IL-6, MMP3) was assayed by RT-qPCR. FIG. 12Fis two bar graphs and FIG. 12G is two bar graphs showing the activity ofK-9 was compared with 3TC in senescent and 3× cells. Senescent cultureswere treated between 12 and 16 weeks (as in FIG. 3B) and 3× cultures for48 hrs (as in panel (a) above). Effects on the expression of IFN-I genes(IFN-α, IRF7, OAS1) and SASP genes (IL-1β IL-6, MMP3) was assessed byRT-qPCR. (FIGS. 12A-G), n=3 independent experiments. (FIGS. 12A-G) Dataare mean±s.d. *P≤0.05, **P≤0.01, unpaired two-sided t-tests.

FIGS. 13A-H show the assessment of p16, L1 ORF1 and pSTAT1 expression insenescent cells and skin specimens from aged humans. FIG. 13A is nineimages showing immunofluorescence (IF) detection of p16 and ORF1 inearly passage, 3× and senescent cells. FIG. 13B is 18 images showingrepresentative images of combinatorial ORF1 and p16 or ORF1p and pSTAT1staining in human dermis. The experiments shown in panels (a, b) wasrepeated three times independently with similar results. FIG. 13C is twographs showing cells were plated on cover slips, stained and quantifiedas described in the Methods. 200 cells in multiple fields were scoredfor each condition. a.f.u., arbitrary fluorescence units. Insets showthe % of cells found in each quadrant. FIG. 13D is a graph and FIG. 13Eis a graph showing abundance of ORF1 and p16 or pSTAT1 cells in humanskin. Skin biopsies were cryosectioned and stained as described in theMethods. 200 dermal fibroblast cells in multiple fields were scored foreach subject. Aggregated data for four subjects (800 cells) are shown.FIG. 13F is a table showing data in (c) and (d) were recalculated toshow the relative abundance of p16+ cells among all cells, and ORF1+cells in the p16+ pool of cells. FIG. 13G is a table showing data in (e)were recalculated as in (f). FIG. 13H is a table showing characteristicsof the human subjects used in the analysis of dermal fibroblasts. Thesespecimens were collected as part of the ongoing Leiden LongevityStudy.¹⁶ The specimens used here were chosen randomly from left overmaterial. The TIF assay¹⁷ relies on a two-parameter (color)visualization of telomeres (using a FISH probe) and immunofluorescentdetection of DNA damage foci (using antibody to 53BP1). Because oflimiting material, it was not possible to combine detection of p16 withTIFs in a three-color experiment.

FIGS. 14A-D show the effects of 3TC or K-9 treatment on L1, p16, IFN-Iand SASP gene expression in mouse tissues. FIGS. 14A-D are each a seriesof eight box plots showing mice at the indicated ages were treated with3TC continuously for two weeks (see also FIGS. 4C, 4E, 15D-F, andMethods). For all conditions the expression of L1 mRNA, p16, threerepresentative IFN-I response genes (Ifn-α, Irf7, Oas1) and threerepresentative SASP genes (II-6, Mmp3, Pai1) were assessed by RT-qPCR.In no instance was expression at 5 months+3TC significantly differentfrom the no drug control; therefore, these data are not shown in thefigure (for all collected data, see Table 7). The box plots show therange of the data (whiskers), 25th and 75 percentiles (box), means(dashed line), and medians (solid line). Each point represents oneanimal. FIG. 14A, Visceral white adipose, male mice. 5 months, n=8animals; 26 months, n=12 animals; 26 months+3TC, n=12 animals. FIG. 14B,Visceral white adipose, female mice. 5 months, n=8 animals; 26 months,n=12 animals; 26 months+3TC, n=12 animals. FIG. 14C, Liver, male mice. 5months, n=8 animals; 26 months, n=10 animals; 26 months+3TC, n=10animals. FIG. 14D, Mice at the age of 26 months were treated with K-9 or3TC in drinking water for two weeks and analyzed by RT-qPCR as above.NT, not treated. Visceral white adipose, male mice, n=7 animals for eachgroup. Data are mean±s.d. *P≤0.05, **P≤0.01, 1-way ANOVA with Tukey'smultiple comparisons test.

FIGS. 15A-G show the combinatorial assessment of senescence, IFN-I, SASPand L1 markers and effects of 3TC on age-associated phenotypes in mousetissues. FIG. 15A and FIG. 158 are each twelve images showingwhole-mount IF was performed on white adipose of 5 months and 26 monthsold (with and without 2 weeks of 3TC treatment) male mice. In (a), lossof Lamin B1 (senescence marker) was colocalized with IL-6 (SASP marker).In (b), pStat1 (IFN-I marker) was colocalized with Orf1 (L1 marker).FIG. 15C is a table showing quantification of the experiments shown in(a) and (b). Four animals and at least 200 cells per animal were scoredfor each condition. FIG. 15D is three images showing neutral lipids werestained with BODIPY to visualize mature adipocytes in whole-mountpreparations, and macrophages were detected by IF using the F4/80antibody. FIG. 15E is five box plots showing the effects of 2 weeks of3TC treatment on adipogenesis were assessed by measuring mean adipocytesize (left panel), and by RT-qPCR to determine the expression of keyadipogenic genes (right panels; Acaca, acetyl-CoA carboxylase 1; Cebpa,CCAAT/enhancer-binding protein alpha; Fasn, fatty acid synthase; Srebp1,sterol regulatory element-binding protein 1). The box plots show therange of the data (whiskers), 25th and 75 percentiles (box), means(dashed line), and medians (solid line). Adipocyte size (BODIPY-stainedarea) was calculated using CellProfiler aggregated data for 5 animalsand 500 total cells are shown. For RT-qPCR data, each point representsone animal; n=6 animals. FIG. 15F is a box plot showing expression ofthe Ucp1 gene (thermogenin) in brown adipose tissue was determined byRT-qPCR and is represented as in (e). n=5 animals. FIG. 15G is three boxplots showing expression of L1 mRNA was determined by RT-qPCR and isrepresented as in (e). 5 months, n=8 animals; 26 months, n=12 animals;29 months, n=6 animals. (FIGS. 15E-G) Data are mean±s.d. *P≤0.05,**P≤0.01. (FIGS. 15C, 15E left panel, FIGS. 15F, 15G) 1-way ANOVA withTukey's multiple comparisons test, (FIG. 15E right panels) unpairedtwo-sided t-tests.

FIGS. 16A-E show scans of raw immunoblots. FIG. 16A: Panel a, shows Blot1-RB1: 1. EP; 2. SEN (L); 3. OE-SEN; 4. SEN (E); Panel b, shows Blot2-TREX1: 1. EP; 2. SEN (E); 3. SEN (L); and Panel c, shows Blot3-FOXA1: 1. EP; 2. Arrest; 3. SEN (E); 4. SEN (L). FIG. 16B: Panel d,shows Blot 4-RB1: 1. EP; 2. 3×; Panel e, shows Blot 5-TREX1: 1. EP; 2.3×; and Panel f, shows Blot 6-FOXA1: 1. EP; 2. 3×. FIG. 16C: Panel g,shows Blot 7-RB1: 1. SEN (L); 2. shRB1(b); 3. shRB1(a); 4. OE-RB1; Panelh, shows Blot 8-TREX1: 1. SEN (L); 2. shTREX1 (b); 3. shTREX1 (a); 4.OE-TREX1; and Panel i, shows Blot 9-FOXA1: 1. shFOXA1 (a); 2. shFOXA1(b); 3. SEN (L); 4. OE-FOXA1. FIG. 16D: Panel j, shows Blot 10-STAT2: 1.3×; 2. shL1; 3. NRTI; 4. ΔIFNAR; 5. EP; Panel k, shows Blot 11-IRF7: 1.EP; 2. shL1; 3. 3×; 4. shL1; 5. ΔIFNAR; Panel l, shows Blot 12-STAT2: 1.SEN (L); 2. ΔIFNAR; 3. shL1; 4. NRTI; and Panel m, shows Blot 13-IRF7:5. SEN (L); 6. ΔIFNAR; 7. shL1; 8. NRTI. FIG. 16E shows: Panel n, showsBlot 14-p16 (CDKN2A): 1. EP; 2. SEN (E); 3. SEN (L); and Panel o, showsBlot 15-p21 (CDKN1A): 1. EP; 2. SEN (E); 3. SEN (L).

FIGS. 17A-D show the effects of Adefovir and Lamivudine onsenescence-induced increases in L1 sequence abundance, interferon geneexpression, and SASP gene expression. FIG. 17A is three bar graphsdepicting the effects of 5 μM Adefovir and Lamivudine on L1 sequenceabundance (copy number) in three different human fibroblast cell lines:LF1, IMR90, WI38 using qPCR assays. FIG. 17B is two bar graphs depictingthe effects of 5 μM Adefovir and Lamivudine on interferon geneexpression of two interferon genes (IFN-α and IFN-β1) in two cell lines(LF1 and IMR90). FIG. 17C is two bar graphs depicting the effects of 5μM Adefovir and Lamivudine on two SASP genes (IL-6 and MMP3) in the LF1cell line. FIG. 17D is two bar graphs depicting the effects of higherdoses of Adefovir and Lamivudine (10 μM and 50 μM) on interferon geneexpression (IFN-α and IFN-β1) in the LF1 cell line.

FIGS. 18A and 188 are each eight dose response curves showing theinhibition of mouse L1 activity with eight RTI compounds: Lamivudine(3TC); Stavudine; Emtricitabine; Apricitabine; Tenofovir Disiproxil;Censavudine; Elvucitabine; and Tenofovir. A dose-response was obtainedon the retrotransposition activity of active mouse LINE-1 using HeLacells in a first experiment (FIG. 18A) and in a second independentexperiment (FIG. 18B).

FIG. 19A is three dose response curves and FIG. 19B is two dose responsecurves showing the inhibition of human L1 activity with three RTIcompounds: Lamivudine (3TC); Censavudine; and Elvucitabine. Adose-response was obtained on the retrotransposition activity of activehuman LINE-1 using HeLa cells in a first experiment using Lamivudine(3TC); Censavudine; and Elvucitabine (FIG. 19A); and in a secondexperiment using Lamivudine (3TC) and Elvucitabine (FIG. 19B).

FIG. 20 is nine dose response curves showing the cell viability of HeLacells following treatment with nine different RTI compounds: Lamivudine(3TC); Stavudine; Emtricitabine; Apricitabine; Tenofovir Disiproxil;Censavudine; Elvucitabine; Tenofovir; and Staurosporine.

DETAILED DESCRIPTION OF THE INVENTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention.

The following description of particular aspect(s) is merely exemplary innature and is in no way intended to limit the scope of the invention,its application, or uses, which may, of course, vary. The invention isdescribed with relation to the non-limiting definitions and terminologyincluded herein. These definitions and terminology are not designed tofunction as a limitation on the scope or practice of the invention butare presented for illustrative and descriptive purposes only. While thecompositions or processes are described as using specific materials oran order of individual steps, it is appreciated that materials or stepsmay be interchangeable such that the description of the invention mayinclude multiple parts or steps arranged in many ways as is readilyappreciated by one of skill in the art.

Definitions

The definitions of certain terms as used in this specification and theappended claims are provided below. Unless defined otherwise, alltechnical and scientific terms used herein generally have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

The term “approximately” or “about” in reference to a value or parameterare generally taken to include numbers that fall within a range of 5%,10%, 15%, or 20% in either direction (greater than or less than) of thenumber unless otherwise stated or otherwise evident from the context(except where such number would be less than 0% or exceed 100% of apossible value). As used herein, reference to “approximately” or “about”a value or parameter includes (and describes) embodiments that aredirected to that value or parameter. For example, description referringto “about X” includes description of “X”.

As used herein, the term “or” means “and/or.” The term “and/or” as usedin a phrase such as “A and/or B” herein is intended to include both Aand B; A or B; A (alone); and B (alone). Likewise, the term “and/or” asused in a phrase such as “A, B, and/or C” is intended to encompass eachof the following embodiments: A, B, and C; A, B, or C; A or C; A or B; Bor C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.It is also understood that wherever embodiments are described hereinwith the language “consisting essentially of” otherwise analogousembodiments described in terms of “consisting of” are also provided.

It is to be appreciated that certain features of the invention whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the invention that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, reference to values statedin ranges include each and every value within that range.

The term “subject” refers to a mammal, including but not limited to adog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate.Subjects can be house pets (e.g., dogs, cats), agricultural stockanimals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals(e.g., mice, rats, rabbits, etc.), but are not so limited. Subjectsinclude human subjects. The human subject may be a pediatric, adult, ora geriatric subject. The human subject may be of either sex.

The terms “effective amount” and “therapeutically-effective amount”include an amount sufficient to prevent or ameliorate a manifestation ofdisease or medical condition, such as an age-associated disorder. Itwill be appreciated that there will be many ways known in the art todetermine the effective amount for a given application. For example, thepharmacological methods for dosage determination may be used in thetherapeutic context. In the context of therapeutic or prophylacticapplications, the amount of a composition administered to the subjectwill depend on the type and severity of the disease and on thecharacteristics of the subject, such as general health, age, sex, bodyweight and tolerance to drugs. It will also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions can also be administered in combination with one or moreadditional therapeutic compounds.

As used herein, the terms “treating” or “treatment” or “to treat” or“alleviating” or “to alleviate” refer to both (1) therapeutic measuresthat cure, slow down, lessen symptoms of, and/or halt progression of adiagnosed disease or infection and (2) prophylactic or preventativemeasures that prevent or slow the development of a disease or infection.

As used herein, the term “long-term” administration means that thetherapeutic agent or drug is administered for a period of at least 12weeks. This includes that the therapeutic agent or drug is administeredsuch that it is effective over, or for, a period of at least 12 weeksand does not necessarily imply that the administration itself takesplace for 12 weeks, e.g., if sustained release compositions or longacting therapeutic agent or drug is used. Thus, the subject is treatedfor a period of at least 12 weeks. In many cases, long-termadministration is for at least 4, 5, 6, 7, 8, 9 months or more, or forat least 1, 2, 3, 5, 7 or 10 years, or more.

As used herein, the term “age-related inflammation” (or “age-associatedinflammation”) is an inflammation, typically a chronic, particularly achronic systemic inflammation which occurs with increasing age. Suchinflammation may be observed above the age of 30, 35 or 40 but typicallyis seen in subjects aged 45, 50, 55 or 60 or more. In many cases thismay be a low-level inflammation.

As used herein, the term “chronic inflammation” means an inflammation(e.g., an inflammatory condition) that is of persistent or prolongedduration in the body of a subject. Generally speaking, this means aninflammatory response or condition of duration of 20, 25 or 30 days ormore or 1 month or more, more particular of at least 2 or 3 months ormore. Chronic inflammation leads to a progressive shift in the type ofcells present at the site of inflammation. Chronic inflammation may be afactor in the development of a number of diseases or disorders,including particularly degenerative diseases, or diseases or conditionsassociated with loss of youthful function or aging.

As used herein, the term “systemic inflammation” is inflammation whichis not confined to a particular tissue or site or location in the body.The inflammation may be generalized throughout the body. Systemicinflammation typically involves the endothelium and other organ systems.

As used herein, the term “low-level inflammation” (which term is usedherein as synonymous with “low-grade inflammation”) is characterized bya 2- to threefold increase in the systemic concentrations of cytokinessuch as TNFα, IL-6, and CRP, e.g., as measured in the plasma or serum.The increase may be relative to, or as compared with, normalconcentrations or reference concentrations, for example concentrationsas determined in a particular reference cohort or population ofsubjects, e.g., young subjects (e.g., young adults) or healthy subjects,for example subjects who are not suffering from any disease orcondition, including any inflammatory disease, or who do not haveinflammation. The increase may also be relative to the level ofconcentration in a subject prior to development of the inflammation.Low-level inflammation may be observed in the absence of overt signs orsymptoms of disease. Thus, low-level inflammation may be sub-clinicalinflammation. Alternatively, a subject with low-level inflammation maynot have a clinically diagnosed condition or disease but may exhibitcertain signs or symptoms of an inflammatory response or inflammatorycondition. In other words, there may be signs or symptoms of the effectof inflammation in the body, but this may not yet have progressed to anovert or recognized disease.

As used herein, the term “cancer inflammation” is inflammation thatoccurs in the context of cancer and may alternatively be defined as“cancer-associated inflammation”. Inflammation has been identified as ahallmark of cancer and may be necessary for tumorigenesis andmaintenance of the cancer state. Cancer symptoms are associated withinflammation. Thus, a subject with cancer may have or exhibitinflammation, which can be a low-level or peripheral inflammation asdiscussed above, and in particular a chronic or systemic inflammation asdiscussed above.

As used herein the term “pathophysiological L1-associated process”refers to a disordered physiological process relating to aberrant LINE-1(L1) retrotransposition activity. See, e.g., Suarez et al., DevNeurobiol 78:434-455 (2018); Saleh et al, (2019) Front. Neurol. 10:894.doi: 10.3389/fneur.2019.00894. Zhao et al., PLoS Genet 15(4): e1008043.https://doi.org/10.1371/journal.pgen.1008043; Bundo et al., Neuron81:306-313 (2014).

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure belongs. It willbe further understood that terms such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

Pharmaceutical Compostions

The compositions and methods of the present invention may be utilized totreat an individual in need thereof. In certain embodiments, theindividual is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the invention and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In preferred embodiments, when suchpharmaceutical compositions are for human administration, particularlyfor invasive routes of administration (i.e., routes, such as injectionor implantation, that circumvent transport or diffusion through anepithelial barrier), the aqueous solution is pyrogen-free, orsubstantially pyrogen-free. The excipients can be chosen, for example,to effect delayed release of an agent or to selectively target one ormore cells, tissues or organs. The pharmaceutical composition can be indosage unit form such as tablet, capsule (including sprinkle capsule andgelatin capsule), granule, lyophile for reconstitution, powder,solution, syrup, suppository, injection or the like. The composition canalso be present in a transdermal delivery system, e.g., a skin patch.The composition can also be present in a solution suitable for topicaladministration, such as a lotion, cream, or ointment.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a self-emulsifying drug deliverysystem or a self-micro emulsifying drug delivery system. Thepharmaceutical composition (preparation) also can be a liposome or otherpolymer matrix, which can have incorporated therein, for example, acompound of the invention. Liposomes, for example, which comprisephospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); subcutaneously; transdermally (for example as a patchapplied to the skin); and topically (for example, as a cream, ointmentor spray applied to the skin). The compound may also be formulated forinhalation. In certain embodiments, a compound may be simply dissolvedor suspended in sterile water. Details of appropriate routes ofadministration and compositions suitable for same can be found in, forexample, U.S. Pat. Nos. 6,110,973; 5,763,493; 5,731,000; 5,541,231;5,427,798; 5,358,970; and 4,172,896, as well as in patents citedtherein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration (capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragées, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropyl methyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragées, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropyl methyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,micro-emulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, con,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the active compound in theproper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intraocular (such as intravitreal),intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal andintrasternal injection and infusion. Pharmaceutical compositionssuitable for parenteral administration comprise one or more activecompounds in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Examples of suitable aqueous and nonaqueous carriersthat may be employed in the pharmaceutical compositions of the inventioninclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants. Proper fluidity can be maintained, for example, by the useof coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically-acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinaceous biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. By “therapeutically effective amount” ismeant the concentration of a compound that is sufficient to elicit thedesired therapeutic effect. It is generally understood that theeffective amount of the compound will vary according to the weight, sex,age, and medical history of the subject. Other factors which influencethe effective amount may include, but are not limited to, the severityof the patient's condition, the disorder being treated, the stability ofthe compound, and, if desired, another type of therapeutic agent beingadministered with the compound of the invention. A larger total dose canbe delivered by multiple administrations of the agent Methods todetermine efficacy and dosage are known to those skilled in the art.¹⁸

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In other embodiments, the active compound will be administeredonce daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans; and other mammals such as equinesbovine, porcine, sheep, feline, and canine; poultry; and pets ingeneral.

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent.

The present disclosure includes the use of pharmaceutically acceptablesalts of compounds of the invention in the compositions and methods ofthe present invention. In certain embodiments, contemplated salts of theinvention include, but are not limited to, alkyl, dialkyl, trialkyl ortetra-alkyl ammonium salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts. In certain embodiments, contemplated salts of theinvention include, but are not limited to, 1-hydroxy-2-naphthoic acid,2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaricacid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid,adipic acid, I-ascorbic acid, I-aspartic acid, benzenesulfonic acid,benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capricacid (decanoic acid), caproic acid (hexanoic acid), caprylic acid(octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, formic acid, fumaric acid, galactaric acid, gentisic acid,d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid,glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid,hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid,lactobionic acid, lauric acid, maleic acid, I-malic acid, malonic acid,mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid,oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionicacid, I-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid,succinic acid, sulfuric acid, I-tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acidsalts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, and the like. Mixtures of such solvates can also beprepared. The source of such solvate can be from the solvent ofcrystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Role of Cellular Senescence in Aging and Age-Associated Diseases

FIG. 5 provides a flow chart outlining the molecular pathway of cellularsenescence leading to age-associate, sterile inflammation, which aredescribed in further detail below.

Upregulation of Retrotransposable Elements (RTEs)

RTEs have been known to be activated transcriptionally in senescentcells¹⁹ and in some mouse tissues.²⁰ Three regulators have beenpreviously shown to be involved with regulation of L1: FOXA1, RB, TREX1.FOXA1 was reported to be upregulated in senescent cells²¹ and to bind tothe L1 promoter.²² RB has been reported to represses L1 elements.²³ Thecomplete loss of TREX1 (germline deletion) has been reported to lead toan autoimmune disease (Aicardi-Gutierrez syndrome, AGS), which isassociated with L1 activation.²⁴

Although these three regulators had been reported to have a role in theregulation of L1, the data in the present disclosure provides the firstdemonstration that the misregulation of the combination of these threefactors is sufficient to allow activation of endogenous L1 elements innormal cells. See, Example 2. Moreover, the present disclosure providesthe first description of late senescence as a discrete temporal, andhitherto unknown, stage of senescence, and one that is characterized bythe upregulation of L1 elements and triggering on an IFN-I response.See, Example 1.

Accumulation of Cytoplasmic L1 cDNA

The existence of cytoplasmic L1 cDNA has been known. One source has beenshown to be from mitochondria.²⁵ The nuclei has been reported to “leak”chromosomal DNA into the cytoplasm in senescent cells.²⁶ These weresubsequently termed “cytoplasmic chromatin fragments” (CCF)²⁷ and laterdescribed in senescent cells.²⁸ However, none of these reports mentionRTEs and L1s as components of CCF.

The data in the present disclosure not only show that L1 DNA sequencesare found in the cytoplasm in senescent cells, but also that they areenriched relative to nuclear DNA sequences. See, Example 3. As such, thepreviously-reported simple extrusion of bulk chromosomal DNA from thenucleus into the cytoplasm cannot explain the enrichment of L1 sequencesobserved herein.

Although it had been previously reported L1 DNA was enriched in TREX1null cells (AGS)²⁹ and that the L1 cDNA accumulating in TREX1 knockoutcells was cytoplasmic in localization,³⁰ AGS in a rare autoimmunedisease that is not age-associated. In contrast, the data in the presentdisclosure generalizes the presence of cytoplasmic L1 DNA to cellularsenescence, one of the major driver of aging and aging-associateddiseases.

Induction of IFN-I and Crosstalk to Immune System, Reinforcement of SASP

Cytoplasmic DNA (and CCF in particular) has been previously reported tobe recognized by the cGAS/STING sensor pathway, which consequentlypromotes an inflammatory state.³¹ In the context of cellular senescence,this pro-inflammatory state is known as the senescence-associatedsecretory phenotype (SASP). These reports showed that SASP was at leastin part dependent on CCF, since knockdown of cGAS/STING pathwaycomponents reduced SASP. The IFN-I response was also reported to be apart of this pro-inflammatory cascade.³² As noted above, none of theseprevious reports implicated L1 elements in the promotion of SASP by CCF.

The data in the present disclosure establish that, not only does L1 DNAcomprise a significant proportion of CCF that is enriched in cytoplasmicDNA relative to nuclear sequences, but it is also functionally relevantfor the promotion of SASP. In particular, the data in the presentdisclosure show that decreasing the amount of cytoplasmic L1 DNA, eitherby shRNA to L1 or by blocking L1 reverse transcription with RTI drugs,reduced both the IFN-I response and SASP in senescent cells.Importantly, the data in the present disclosure represent the firstevidence that RTI treatment can effectively reverse both IFN-I and theproinflammatory SASP after they are fully established in senescentcells. See, Examples 3 and 4.

All prior work relevant to therapy has been limited to interfering withthe cGAS/STING sensor pathway, for example showing that shRNAs againstcomponents of the cGAS/STING pathway downregulated the IFN-I responseand SASP.^(33,34) Although one could envision using small molecularinhibitors of the cGAS/STING pathway to downregulate SASP, suchtreatments would result in increased sensitivity to viral, bacterial andother pathogen infections.

The approach of the present invention of targeting the synthesis of L1DNA with RTI drugs goes to the root of the problem because it targetsthe prime causative agent (L1 DNA itself), as opposed to downstreamprocessing events, such as the cGAS/STING sensor pathway, or evenfurther downstream interferon or immune signaling components. It hasbeen appreciated in the present invention that all of these downstreamcomponents have essential cellular functions, and hence targeting themwould compromise, in some aspect, normal physiological processes. On theother hand, the L1 DNA is a unique “non-self” component, whosepharmacological targeting would only be compromised by “off-target”effects.

In recent years, it has become clear that cellular senescence is one ofthe major drivers of organismal aging and aging-associated diseases.³⁵Hence, there is considerable interest in “seno-therapies” to block thedeleterious effects of senescent cells.³⁶ The major effort has beendirected at “senolytic” drugs that selectively kill (and hence remove)senescent cells from tissues. “Senomorphics” have been classified assmall molecules that suppress senescent phenotypes without cell killing.We prefer to refer to such drugs as “senostatic” drugs to emphasize thattheir main effect is to halt, or block the harmful effects of senescentcells, in particular the SASP.

The data of the present disclosure demonstrate that, in human cells andmouse models, RTIs are senostatic drugs that reverse the SASP ofsenescent cells and hence alleviate the age-associated proinflammatorystate. The data of the present disclosure also describe which specificRTIs, and which doses, are particularly effective in reversing SASP. Thebroad efficacy of RTIs as senostatic drugs that can treat multipleage-associated conditions has not been previously described in the art.

Promotion of Age-Associated, “Sterile” Inflammation

Sterile inflammation, also known as inflammaging, is a hallmark of agingand a contributing factor to many age-related diseases.^(37,38) The dataof the present disclosure indicate that activation of L1 elements (andpossibly other RTEs) promotes inflammaging, and that the L1 RT is arelevant target for the treatment of age-associated inflammation anddisorders.

The data of the present disclosure provide specific examples, in agedmice, of which age-associated pathologies can be reversed, or at leastdownregulated, by administration of RTIs. For example, the NRTILamivudine (aka 3TC or Epivir) was shown to reverse or downregulate:

-   -   Panel of IFN-I and SASP markers, measured by RT-qPCR in multiple        tissues;    -   Infiltration of macrophages into white adipose and kidney        tissues measured by IF microscopy;    -   Muscle atrophy measured by muscle fiber diameter;    -   Kidney glomerulosclerosis measured by pathological evaluation of        PAS-stained sections;    -   Adipocyte atrophy measured microscopically by cell size and by        RT-qPCR analysis of key adipogenic genes; and    -   Thermogenesis measured by RT-qPCR analysis of Ucp1 expression.

Accordingly, the present invention provides that RTIs can be used as“senostatic” drugs that are able to halt, or block the harmful effectsof senescent cells, in particular the SASP, and prevent or reverseage-related inflammation and disorders.

Nucleoside Reverse Transcriptase Inhibitors (NRTIs)

NRTIs are active inhibitors of reverse transcriptase found inretroviruses such as the human immunodeficiency virus (HIV). Thedifferent nucleoside reverse transcriptase inhibitors may be activateddifferently but they have the same mechanism of action. NRTIs areactivated generally by phosphorylation to the triphosphate form bycellular enzymes. It then competes with cellular triphosphates, whichare substrates for proviral DNA by viral reverse transcriptase. NRTIswere the first type of drugs available for the treatment of humanimmunodeficiency virus (HIV infection) and acquired immune deficiencysyndrome (AIDS).

Table 8 provides a list of common approved NRTI drugs or NRTIcombination drugs used for the treatment of HIV infection and AIDS. Asdescribed above, according to the methods of the present invention,NRTIs can be used as “senostatic” drugs that are able to halt, or blockthe harmful effects of senescent cells, in particular the SASP, andprevent or reverse age-related inflammation and disorders.

NRTI drugs that can be used in the methods of the present inventioninclude, but are not limited to: Amdoxovir, Apricitabine (ATC), ATRIPLA®(efavirenz/emtricitabine/tenofovir disoproxil), BARACLUDE® (entecavir;ETV), BIKTARVY® (bictegravir/emtricitabine/tenofovir alafenamide),Censavudine (INN; BMS-986001; OBP-601; festinavir), COMBIVIR™(zidovudine/lamivudine), COVIRACIL™ (emtricitabine; FTC), DAPD/DXG(active metabolite of DAPD-2,6-diaminopurine dioxolane), DESCOVY®(emtricitabine/tenofovir alafenamide), D-D4FC (Dexelvucitabine;Reverset; INCB-8721; DPC 817), dOTC (2′-Deoxy-3′-Oxa-4′-Thiocytidine;BCH-10652), Elvucitabine, EMTRIVA™ (emtricitabine), EPIVIR™ (lamivudine;3TC), EFdA (4′-Ethynyl-2-fluoro-2′-deoxyadenosine; MK-8591), EVIPLERA™(rilpivirine/emtricitabine/tenofovir disoproxil), GENVOYA®(elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide), HIVID™(zalcitabine; ddC), KIVEXA™ (abacavir/lamivudine), LODENOSINE™ (F-ddA),ODEFSEY® (rilpivirine/tenofovir alafenamide/emtricitabine), PREVEON®(adefovir dipivoxil), Racivir (RCV; (+/−)-Emtricitabine), RETROVIR™(zidovudine; ZDV; azidothymidine; AZT), Stampidine, STRIBILD®(Elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil), TENOFOVIR™(TDF, bis-POC PMPA), TRIUMEQ® (dolutegravir/abacavir/lamivudine),TRIZIVIR™ (abacavir/lamivudine/zidovudine), TRUVADA®(emtricitabine/tenofovir disoproxil), VEMLIDY® (tenofovir alafenamide;TAF), VIDEX™ (didanosinel ddl), VIREAD™ (tenofovir disoproxil), ZIAGEN™(abacavir; 159U89), and ZERIT™ (stavudine; d4T).

At the doses used for HIV/AIDS therapy, these drugs can cause a widerange of side effects. Common Side Effects of NRTIs include, inter alia,mitochondrial toxicity (associated with inhibition of mitochondrialpolymerase), neuropathy, pancreatitis, hepatic steatosis and lacticacidosis, myelosuppression, symptomatic myopathy, and cardiomyopathy.³⁹Although the NRTIs can be used at the dosage approved for HIV/AIDStreatment, lower dosages can be used for the prevention or treatment ofage-related inflammation and disorders in order to avoid the sideeffects associated with the higher doses. In one embodiment, the dosageused for the prevention or treatment of age-related inflammation anddisorders is half (50%) of the dosage approved for HIV/AIDS treatment(see Table 9). In an alternate embodiment, the dosage used is 75% of thedosage approved for HIV/AIDS treatment. In yet another alternateembodiment, the dosage used is 25% of the dosage approved for HIV/AIDStreatment. In other alternate embodiments, the dosage used is 90%, 80%,70%, 60%, 40%, 30%, 20%, or 10% of the dosage approved for HIV/AIDStreatment. In yet other alternate embodiments, the dosage used is 0.1 to99.5%, 10 to 90%, 20 to 80%, 25 to 75%, 30 to 70%, 40 to 60% or 45 to55% of the dosage approved for HIV/AIDS treatment.

In some embodiments, the subject undergoes long term administration ofone or more RTI drugs, as defined herein. In one embodiment, the subjectis treated for a period of at least 12 weeks. In many cases, long-termadministration is for at least 4, 5, 6, 7, 8, 9 months or more, or forat least 1, 2, 3, 5, 7 or 10 years, or more.

Age-Associated Disorders

Given that cellular senescence is one of the major drivers of organismalaging and aging-associated diseases,⁴⁰ the methods of the presentinvention can be used to prevent or treat disorders or diseases thathave been associated with cellular senescence, in particular in whichthe presence of senescent cells is likely to have a deleterious effect,by the administration of one or more senostatic RTI drugs.

Disorders or diseases that have been associated with cellular senescenceinclude, but are not limited to, Alzheimer's disease,⁴¹ amyotrophiclateral sclerosis (ALS), atherosclerosis,⁴² Huntington's disease, visionloss, hearing loss, peripheral degenerative diseases, cardiovasculardysfunction,⁴³ atherosclerosis, frontotemporal dementia (FTD), multiplesclerosis (MS), Aicardi Goutiere's syndrome, progressive supra nuclearpalsy (PSP), chemotherapy-induced adverse effects (e.g., bone marrowsuppression, cardiotoxicity, cancer recurrence, blood clots, fatigue),⁴⁴hematopoietic stem cell function,⁴⁵ osteoarthritis,⁴⁶ osteoporosis,⁴⁷osteoporosis, Parkinson's disease,⁴⁸ physical function,⁴⁹ pulmonaryfibrosis,⁵⁰ skin aging, wound healing, and/or tissue regeneration.⁵¹

Methods of Treating, Preventing and/or Reversing Age-AssociatedInflammation and Other Diseases or Disorders with RTIs

Provided is a method for treating, preventing and/or reversing apathophysiological L1-associated process, e.g., age-associatedinflammation, in a patient in need thereof by administering a reversetranscriptase inhibitor (RTI) to a patient in need thereof. In someembodiments, the age-associated inflammation may be in a patient havingAlzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease,Huntington's disease, vision loss, hearing loss, peripheral degenerativediseases, and cardiovascular dysfunction. In some embodiments, thedisclosure provides a method for treating, preventing and/or reversingAlzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporaldementia (FTD), progressive supra nuclear palsy (PSP), dementia withLewy bodies (DLB), multi systems atrophy (MSA), corticobasaldegeneration (CDB), mild cognitive impairment (MCI), Parkinson'sdisease, Huntington's disease, Rett Syndrome, schizophrenia, autismspectrum disorder (ADS), vision loss, hearing loss, peripheraldegenerative diseases, cardiovascular dysfunction, and autoimmunedisease.

In one embodiment is provided a method for delaying or reversing theprogression of the underlying pathology of an age-associatedinflammatory disorder, comprising administering to a patient in needthereof a therapeutically effective amount of at least one reversetranscriptase inhibitor (RTI). In some embodiments, the patientexperiences a decrease in one or more symptoms of Alzheimer's diseasecompared to before the first administration of the RTI to the patient.

In another embodiment, provided is a method for preventing the onset ofan age-associated inflammatory disorder in a patient suspected of havingmild cognitive impairment, comprising administering at least one RTI toa patient in need thereof.

In some embodiments, the NRTI is abacavir, lamivudine, zidovudine,emtricitabine, tenofovir disoproxil fumarate, tenofovir alafenamide,didanosine, stavudine, apricitabine, alovudine, dexelvucitabine,amdoxovir, fosalvudine or elvucitabine. In other embodiments, the RTI isabacavir (Ziagen), abacavir/lamivudine (Epzicom),abacavir/lamivudine/zidovudine (Trizivir), lamivudine/zidovudine(Combivir), lamivudine (Epivir), zidovudine (Retrovir),emtricitabine/tenofovir disoproxil fumarate (Truvada), emtricitabine(Emtriva), tenofovir disoproxil fumarate (Viread),emtricitabine/tenofovir alafenamide (Descovy), didanosine (Videx),didanosine extended-release (Videx EC), or stavudine (Zerit). In anotherembodiment, the NRTI is censavudine.

In some embodiments, the at least one RTI is a non-nucleoside reversetranscriptase inhibitor (NNRTI). In some embodiments, the at least oneNRTI is Efavirenz (EFV), Nevirapine (NVP), Delavirdine (DLV), Etravirineor Rilvipirine.

In a further embodiment, the RTI inhibits L1 reverse transcriptaseactivity in a cell, e.g., a brain cell, of the patient.

Where the RTI is an FDA approved drug, the RTI may be administered intherapeutically effective amounts that are approved for therapeutic use.In other embodiments, the amounts effective can be determined with nomore than routine experimentation. For example, amounts effective mayrange from about 1 ng/kg to about 200 mg/kg, about 1 μg/kg to about 100mg/kg, or about 1 mg/kg to about 50 mg/kg. The dosage of a compositioncan be at any dosage including, but not limited to, about 1 μg/kg. Thedosage of a composition may be at any dosage including, but not limitedto, about 1 μg/kg, about 10 μg/kg, about 25 μg/kg, about 50 μg/kg, about75 μg/kg, about 100 μg/kg, about 125 μg/kg, about 150 μg/kg, about 175μg/kg, about 200 μg/kg, about 225 μg/kg, about 250 μg/kg, about 275μg/kg, about 300 μg/kg, about 325 μg/kg, about 350 μg/kg, about 375μg/kg, about 400 μg/kg, about 425 μg/kg, about 450 μg/kg, about 475μg/kg, about 500 μg/kg, about 525 μg/kg, about 550 μg/kg, about 575μg/kg, about 600 μg/kg, about 625 μg/kg, about 650 μg/kg, about 675μg/kg, about 700 μg/kg, about 725 μg/kg, about 750 μg/kg, about 775μg/kg, about 800 μg/kg, about 825 μg/kg, about 850 μg/kg, about 875μg/kg, about 900 μg/kg, about 925 μg/kg, about 950 μg/kg, about 975μg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg,about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40mg/kg, about 45 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg,about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 125 mg/kg, about150 mg/kg, about 175 mg/kg, about 200 mg/kg, or more. In otherembodiments, the dosage is 1 mg-500 mg. In some embodiments, the dosageis 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150mg. These doses may be unitary or divided and may be administered one ormore times per day. The above dosages are exemplary of the average case,but there can be individual instances in which higher or lower dosagesare merited, and such are within the scope of this disclosure. Inpractice, the physician determines therapeutically effective amounts andthe actual dosing regimen that is most suitable for an individualsubject, which can vary with the age, weight, and response of theparticular subject.

The RTI may be administered once, twice or three times per day for 1 dayto the end of life, or for 1 day to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore years, or until the RTI causes unacceptable side effects or is nolonger useful.

The patient is monitored for changes in the symptoms of theage-associated inflammatory disease. In one embodiment, there is areduction in the symptoms. In another embodiment, the symptoms remainabout the same and there is no evidence of progression. In connectionwith Alzheimer's disease, such symptoms include memory loss, misplacingitems, forgetting the names of places or objects, repeating questions,being less flexible, confusion, disorientation, obsessive behavior,compulsive behavior, delusions, aphasia, disturbed sleep, mood swings,depression, anxiety, frustration, agitation, difficulty in performingspatial tasks, agnosia, difficulty with ambulation, weight loss, loss ofspeech, loss of short term memory or loss of long term memory. Methodsfor monitoring and quantifying any change of these symptoms can becarried out by routine methods or by routine experimentation.

In one embodiment, symptoms of mild cognitive impairment and any changein the symptoms of Alzheimer's disease is determined using the criteriaset forth in DSM-5. In another embodiment, symptoms of mild cognitiveimpairment and the any change in the symptoms of Alzheimer's disease isdetermined using the Clinician's Interview-Based Impression of Change(CIBIC-plus). In another embodiment, symptoms of mild cognitiveimpairment and any change in symptoms is determined using theClinician's Interview-Based Impression of Change (CIBIC-plus).

Any change in symptoms may be monitored for 1-36 months or more, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36months.

In another embodiment, the patient is monitored for a change in theunderlying pathology of Alzheimer's disease. In one embodiment, there isa reduction in the underlying pathology. In another embodiment, theunderlying pathology remains about the same and there is no evidence ofprogression.

In some embodiments, any change in the underlying pathology isidentified by detection of a biomarker before and after the RTIadministration. In one embodiment, the biomarker is β-amyloid or Tauprotein. In another embodiment, the biomarker is detected by PETimaging. In another embodiment, the underlying pathology is identifiedby measurement of brain volume before and after the RTI administration.

In some embodiments, the decrease of the underlying pathology isreversed or delayed for at 1-36 months, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the firstadministration of the RTI.

In some embodiments, the patient is also administered at least onesecond therapeutic agent useful for the treatment of the symptoms of anage-associated inflammatory disorder. In one embodiment, the patient isadministered at least one second therapeutic agent useful for thetreatment of Alzheimer's disease. In some embodiments, the at least onesecond therapeutic agent is donepezil, galantamine, rivastigmine, ormemantine. In another embodiment, the at least one second therapeuticagent is an antibody that binds to β-amyloid or Tau protein. In anotherembodiment, the antibody binds to β-amyloid and is bapineuzumab. Inanother embodiment, the antibody binds to Tau protein and is ABBV-8E12.In another embodiment, the at least one second therapeutic agent is avaccine against β-amyloid or Tau protein. In another embodiment, the atleast one second therapeutic agent is an agent that reduces or altersthe brain content of β-amyloid or Tau. In another embodiment, the secondtherapeutic agent reduces or alters the brain content of β-amyloid andis a β-secretase 1 (BACE) inhibitor. In another embodiment, the BACEinhibitor is CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293)or LY2886721. In another embodiment, the second agent reduces or altersthe brain content of β-amyloid or Tau alters the brain content of Tauand is nicotinamide, or MPT0G211.

The at least one RTI and at least one second therapeutic agent may beadministered separately or together as part of a unitary pharmaceuticalcomposition.

When the age-associated inflammatory disorder is ALS, the patient may beadministered at least one second agent useful for the treatment of thesymptoms of ALS. In some embodiments, the at least one second agent isan integrase inhibitor. In some embodiments, the integrase inhibitor israltegravir, curcumin, derivatives of curcumin, chicoric acid,derivatives of chicoric acid, 3,5-dicaffeoylquinic acid, derivatives of3,5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives ofaurintricarboxylic acid, caffeic acid phenethyl ester, derivatives ofcaffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin,quercetin, derivatives of quercetin, S-1360, zintevir (AR-177),L-870812, and L-25 870810, MK-0518, BMS-538158, or GSK364735C.⁵²

The patient may be monitored for improvement of the symptoms of ALS.Such symptoms include one or more of the following: difficulty walkingor doing normal daily activities, tripping and falling, weakness of thelegs, feet or ankles, hand weakness or clumsiness, slurred speech ortrouble swallowing, muscle cramps, twitching in the arms, shoulders ortongue, inappropriate crying, cognitive changes, and behavior changes.

Any change in symptoms may be monitored for 1-36 months or more, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36months. In some embodiments, the decrease of the underlying pathology isreversed or delayed for at 1-36 months, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the firstadministration of the RTI.

Salts, Pharmaceutical Compositions, and Kits

The methods of the present disclosure can be accomplished byadministering an at least one RTI as the neat compound or as apharmaceutical composition. Administration of a pharmaceuticalcomposition, or neat compound of an RTI, can be performed before orafter the clinical diagnosis of a disorder associated withage-associated inflammation. Typically, the pharmaceutical compositionsare sterile, and contain no toxic, carcinogenic, or mutagenic compoundsthat would cause an adverse reaction when administered.

Further provided are kits comprising at least one RTI and, optionally,at least one second therapeutic agent useful for the treatment orprevention of a disorder associated with age-associated inflammation,packaged separately or together, and an insert having instructions forusing these active agents. In one embodiment, the at least one RTI ispackaged alone together with instructions to administered together withthe at least one second therapeutic agent. The at least one RTI and theat least one second therapeutic agent can be administered simultaneouslyor sequentially to achieve the desired effect. In addition, the RTI andthe at least one second therapeutic agent can be administered from asingle composition or two separate compositions.

Examples of the at least one second therapeutic agents useful for thetreatment of Alzheimer's disease that may be in the kit includedonepezil, galantamine, rivastigmine and memantine. Other optionaltherapeutic agents that may be in the kit include an antibody that bindsto β-amyloid or Tau protein. In one embodiment, the antibody binds toβ-amyloid and is bapineuzumab. In another embodiment, the antibody bindsto Tau and is ABBV-8E12.

In another embodiment, the kit may contain least one second therapeuticagent that is a vaccine against β-amyloid or Tau protein.

In another embodiment, the kit may contain at least one secondtherapeutic agent that reduces or alters the brain content of β-amyloidor Tau protein. In some embodiments, the second therapeutic agent thatalters or reduces the brain content of β-amyloid is a β-secretase 1(BACE) inhibitor. In some embodiment, the BACE inhibitor is CTS-21166,verubecestat (MK-8931), lanabecestat (AZD3293) or LY2886721, each ofwhich have been in clinical trials for the treatment of Alzheimer'sdisease.

In another embodiment, the kit may contain a second agent that reducesor alters the brain content of Tau and is nicotinamide, or MPT0G211.

In some embodiments, the patient has ALS and the kit further comprisesat least one second agent useful for the treatment of ALS. In otherembodiments, the RTI is packaged alone together with instructions toadminister at least one second therapeutic agent for the treatment ofALS. In some embodiments, the at least one second therapeutic agent forthe treatment of ALS is edaravone or riluzole.

In some embodiments, the at least one second agent is an integraseinhibitor. In some embodiments, the integrase inhibitor is raltegravir,curcumin, derivatives of curcumin, chicoric acid, derivatives ofchicoric acid, 3,5-dicaffeoylquinic acid, derivatives of3,5dicaffeoylquinic acid, aurintricarboxylic acid, derivatives ofaurintricarboxylic acid, caffeic acid phenethyl ester, derivatives ofcaffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin,quercetin, derivatives of quercetin, S-1360, zintevir (AR-177),L-870812, and L-25 870810, MK-0518, BMS-538158, or GSK364735C.⁵³

The second therapeutic agent is administered in an amount to provide itsdesired therapeutic effect. The effective dosage range for each optionaltherapeutic agent is known in the art, and the optional therapeuticagent is administered to an individual in need thereof within suchestablished ranges.

The present disclosure encompasses the preparation and use of salts ofRTIs. As used herein, a “pharmaceutically acceptable salt” refers tosalts or zwitterionic forms of the RTIs. Salts of RTIs can be preparedduring the final isolation and purification of the compounds orseparately by reacting the compound with a suitable acid. Thepharmaceutically acceptable salts of RTIs can be acid addition saltsformed with pharmaceutically acceptable acids. Examples of acids whichcan be employed to form pharmaceutically acceptable salts includeinorganic acids such as nitric, boric, hydrochloric, hydrobromic,sulfuric, and phosphoric, and organic acids such as oxalic, maleic,succinic, and citric. Non-limiting examples of salts of RTIs include,but are not limited to, the hydrochloride, hydrobromide, hydroiodide,sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogenphosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate,butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate,hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate,maleate, ascorbate, isethionate, salicylate, methanesulfonate,mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3phenylproprionate, picrate, pivalate, propionate, trichloroacetate,trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate, undecanoate, lactate, citrate, tartrate,gluconate, methanesulfonate, ethanedisulfonate, benzene sulfonate, and ptoluenesulfonate salts. In addition, available amino groups present inthe RTIs can be quaternized with methyl, ethyl, propyl, and butylchlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamylsulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, andiodides; and benzyl and phenethyl bromides. In light of the foregoing,any reference to RTIs appearing herein is intended to include the RTIsas well as pharmaceutically acceptable salts, hydrates, or solvatesthereof.

The present disclosure encompasses the preparation and use of solvatesof RTIs. Solvates typically do not significantly alter the physiologicalactivity or toxicity of the compounds, and as such may function aspharmacological equivalents. The term “solvate” as used herein is acombination, physical association and/or solvation of a compound of thepresent disclosure with a solvent molecule such as, e.g., a disolvate,monosolvate or hemisolvate, where the ratio of solvent molecule tocompound of the present disclosure is about 2:1, about 1:1 or about 1:2,respectively. This physical association involves varying degrees ofionic and covalent bonding, including hydrogen bonding. In certaininstances, the solvate can be isolated, such as when one or more solventmolecules are incorporated into the crystal lattice of a crystallinesolid. Thus, “solvate” encompasses both solution-phase and isolatablesolvates. RTIs can be present as solvated forms with a pharmaceuticallyacceptable solvent, such as water, methanol, and ethanol, and it isintended that the disclosure includes both solvated and unsolvated formsof RTIs. One type of solvate is a hydrate. A “hydrate” relates to aparticular subgroup of solvates where the solvent molecule is water.Solvates typically can function as pharmacological equivalents.Preparation of solvates is known in the art. See, e.g., Caira et al.(2004),⁵⁴ which describes the preparation of solvates of fluconazolewith ethyl acetate and with water. Similar preparation of solvates,hemisolvates, hydrates, and the like are described by Van Tonder et al.(2004)⁵⁵ and Bingham et al. (2001).⁵⁶ A typical, non-limiting, processof preparing a solvate would involve dissolving the at least one RTI orat least one second therapeutic agent in a desired solvent (organic,water, or a mixture thereof) at temperatures above 20° C. to about 25°C., then cooling the solution at a rate sufficient to form crystals, andisolating the crystals by known methods, e.g., filtration. Analyticaltechniques such as infrared spectroscopy can be used to confirm thepresence of the solvate in a crystal of the solvate.

The at least one RTI and at least one second therapeutic agent typicallyare administered in admixture with a pharmaceutical carrier to give apharmaceutical composition selected with regard to the intended route ofadministration and standard pharmaceutical practice. Pharmaceuticalcompositions for use in accordance with the present disclosure areformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and/or auxiliaries thatfacilitate processing of the at least one RTI and at least one secondtherapeutic agent.

These pharmaceutical compositions can be manufactured, for example, byconventional mixing, dissolving, granulating, dragee-making,emulsifying, encapsulating, entrapping, or lyophilizing processes.Proper formulation is dependent upon the route of administration chosen.When a therapeutically effective amount of the at least one RTI and/orat least one second therapeutic agent is administered orally, thecomposition typically is in the form of a tablet, capsule, powder,solution, or elixir. When administered in tablet form, the compositionadditionally can contain a solid carrier, such as a gelatin or anadjuvant. The tablet, capsule, and powder contain about 0.01% to about95%, and preferably from about 1% to about 50%, of the at least one RTIand at least one second therapeutic agent. When administered in liquidform, a liquid carrier, such as water, petroleum, or oils of animal orplant origin, can be added. The liquid form of the composition canfurther contain physiological saline solution, dextrose or othersaccharide solutions, or glycols. When administered in liquid form, thecomposition contains about 0.1% to about 90%, and preferably about 1% toabout 50%, by weight, of the at least one RTI and at least one secondtherapeutic agent.

When a therapeutically effective amount of the at least one RTI and atleast one second therapeutic agent is administered by intravenous,cutaneous, or subcutaneous injection, the composition is in the form ofa pyrogen-free, parenterally acceptable aqueous solution. Thepreparation of such parenterally acceptable solutions, having due regardto pH, isotonicity, stability, and the like, is within the skill in theart. A preferred composition for intravenous, cutaneous, or subcutaneousinjection typically contains, an isotonic vehicle.

The at least one RTI and at least one second therapeutic agent can bereadily combined with pharmaceutically acceptable carriers well-known inthe art. Standard pharmaceutical carriers are described in Remington'sPharmaceutical Sciences.⁵⁷ Such carriers enable the active agents to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject tobe treated. Pharmaceutical preparations for oral use can be obtained byadding the at least one RTI and/or the at least one second therapeuticagent to a solid excipient, optionally grinding the resulting mixture,and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, for example, fillers and cellulose preparations. Ifdesired, disintegrating agents can be added.

The at least one RTI and at least one second therapeutic agent can beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampules or in multidosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active agent in water-soluble form.Additionally, suspensions of the at least one RTI and at least onesecond therapeutic agent can be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fatty oilsor synthetic fatty acid esters. Aqueous injection suspensions cancontain substances which increase the viscosity of the suspension.Optionally, the suspension also can contain suitable stabilizers oragents that increase the solubility of the compounds and allow for thepreparation of highly concentrated solutions. Alternatively, a presentcomposition can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The at least one RTI and at least one second therapeutic agent also canbe formulated in rectal compositions, such as suppositories or retentionenemas, e.g., containing conventional suppository bases. In addition tothe formulations described previously, the at least one RTI and at leastone second therapeutic agent also can be formulated as a depotpreparation. Such long-acting formulations can be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the at least one RTI and atleast one second therapeutic agent can be formulated with suitablepolymeric or hydrophobic materials (for example, as an emulsion in anacceptable oil) or ion exchange resins.

In particular, the at least one RTI and at least one second therapeuticagent can be administered orally, buccally, or sublingually in the formof tablets containing excipients, such as starch or lactose, or incapsules or ovules, either alone or in admixture with excipients, or inthe form of elixirs or suspensions containing flavoring or coloringagents. Such liquid preparations can be prepared with pharmaceuticallyacceptable additives, such as suspending agents. The at least one RTIand at least one second therapeutic agent also can be injectedparenterally, for example, intravenously, intramuscularly,subcutaneously, or intracoronarily. For parenteral administration, theat least one RTI and at least one second therapeutic agent are typicallyused in the form of a sterile aqueous solution which can contain othersubstances, for example, salts or monosaccharides, such as mannitol orglucose, to make the solution isotonic with blood.

Islatravir

In some embodiments, provided is a method for treating, preventingand/or reversing Alzheimer's disease, amyotrophic lateral sclerosis(ALS), frontotemporal dementia (FTD), progressive supra nuclear palsy(PSP), dementia with Lewy bodies (DLB), multi systems atrophy (MSA),corticobasal degeneration (CDB), mild cognitive impairment (MCI),Parkinson's disease, Huntington's disease, vision loss, hearing loss,peripheral degenerative diseases, cardiovascular dysfunction, andautoimmune disease by administering to a patient in need thereof atherapeutically effective amount of islatravir (also known as EDdA,MK-8591 or 2′-deoxy-4′-ethynyl-2-fluoroadenosine). Islatravir and itsmethod of synthesis is described in U.S. Pat. No. 7,625,877. Thechemical structure of islatravir is:

In some embodiments, islatravir is administered daily in an amount thatranges from about 0.1 mg to about 20 mg, e.g., about 0.2 mg to about 15mg, e.g., about 1 mg to about 10 mg, e.g. 0.1 mg, 0.15 mg, 0.2 mg, 0.25mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg,0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg,6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In some embodiments, islatravir isadministered daily in an amount of 0.25, 0.5 or 0.75 mg.

In some embodiments, islatravir is administered monthly in an amountfrom 50-150 mg, e.g., 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg,85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130mg, 135 mg, 140 mg, 145 mg, or 150 mg.

In some embodiments, islatravir is administered by continuous releasefrom an implant. In some embodiments, the implant comprises 30 to 80 mgislatravir. In some embodiments, the implant comprises 30 mg, 35 mg, 40mg, 45 mg, 50 mg, 51 mg, 52 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 70 mg, 75 mg, or 80 mg. Insome embodiments, islatravir is administered by continuous release froman implant containing 54 or 62 mg islatravir.

In some embodiments, islatravir is administered to a patient is (a) notinfected with the HIV virus, (b) is not suspected of being infected withthe HIV virus, and/or (c) is not being treated to prevent infection withthe HIV virus.

In one embodiment is provided a method for delaying or reversing theprogression of the underlying pathology of Alzheimer's disease,comprising administering to a patient in need thereof a therapeuticallyeffective amount of islatravir. In some embodiments, the patientexperiences a decrease in one or more symptoms of Alzheimer's diseasecompared to before the first administration of islatravir to thepatient.

In another embodiment, provided is a method for preventing the onset ofAlzheimer's disease in a patient suspected of having mild cognitiveimpairment, comprising administering islatravir to a patient in needthereof.

In some embodiments, the patient is monitored for changes in thesymptoms of the disorder after the first administration of islatravir.In one embodiment, there is a reduction in the symptoms. In anotherembodiment, the symptoms remain about the same and there is no evidenceof progression of the disorder. In connection with Alzheimer's disease,such symptoms include memory loss, misplacing items, forgetting thenames of places or objects, repeating questions, being less flexible,confusion, disorientation, obsessive behavior, compulsive behavior,delusions, aphasia, disturbed sleep, mood swings, depression, anxiety,frustration, agitation, difficulty in performing spatial tasks, agnosia,difficulty with ambulation, weight loss, loss of speech, loss of shortterm memory or loss of long term memory. Methods for monitoring andquantifying any change of these symptoms can be carried out by routinemethods or by routine experimentation.

In one embodiment, symptoms of mild cognitive impairment and any changein the symptoms of Alzheimer's disease is determined using the criteriaset forth in the Diagnostic and Statistical Manual of Mental Disorders,Fifth Edition (DSM-5). In another embodiment, symptoms of mild cognitiveimpairment and the any change in the symptoms of Alzheimer's disease isdetermined using the Clinician's Interview-Based Impression of Change(CIBIC-plus). In another embodiment, symptoms of mild cognitiveimpairment and any change in symptoms is determined using theClinician's Interview-Based Impression of Change (CIBIC-plus).

Any change in symptoms may be monitored for 1-36 months or more, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or36 months.

In another embodiment, the patient is monitored for a change in theunderlying pathology of Alzheimer's disease. In one embodiment, there isa reduction in the underlying pathology. In another embodiment, theunderlying pathology remains about the same and there is no evidence ofprogression.

In some embodiments, any change in the underlying pathology isidentified by detection of a biomarker before and after islatraviradministration. In one embodiment, the biomarker is β-amyloid or Tauprotein. In another embodiment, the biomarker is detected by PETimaging. In another embodiment, the underlying pathology is identifiedby measurement of brain volume before and after islatraviradministration.

In some embodiments, the decrease of the underlying pathology isreversed or delayed for 1-36 months, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the firstadministration of islatravir.

In some embodiments, the patient is also administered at least onesecond therapeutic agent useful for the treatment of the symptoms ofAlzheimer's disease, ALS, FTD, PSP, DLB, MSA, CDB, MCI, Parkinson'sdisease, Huntington's disease, vision loss, hearing loss, peripheraldegenerative diseases, cardiovascular dysfunction, or autoimmunedisease. In one embodiment, the patient is administered at least onesecond therapeutic agent useful for the treatment of Alzheimer'sdisease. In some embodiments, the at least one second therapeutic agentis donepezil, galantamine, rivastigmine, or memantine. In anotherembodiment, the at least one second therapeutic agent is an antibodythat binds to β-amyloid or Tau protein. In another embodiment, theantibody binds to β-amyloid and is bapineuzumab. In another embodiment,the antibody binds to Tau protein and is ABBV-8E12. In anotherembodiment, the at least one second therapeutic agent is a vaccineagainst s-amyloid or Tau protein. In another embodiment, the at leastone second therapeutic agent is an agent that reduces or alters thebrain content of β-amyloid or Tau. In another embodiment, the secondtherapeutic agent reduces or alters the brain content of β-amyloid andis a β-secretase 1 (BACE) inhibitor. In another embodiment, the BACEinhibitor is CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293)or LY2886721. In another embodiment, the second agent reduces or altersthe brain content of β-amyloid or Tau alters the brain content of Tauand is nicotinamide, or MPT0G211.

Islatravir and at least one second therapeutic agent may be administeredseparately or together as part of a unitary pharmaceutical composition.

When the age-associated disorder is ALS, the patient may be administeredat least one second agent useful for the treatment of the symptoms ofALS. In some embodiments, the at least one second agent is an integraseinhibitor. In some embodiments, the integrase inhibitor is raltegravir,curcumin, derivatives of curcumin, chicoric acid, derivatives ofchicoric acid, 3,5-dicaffeoylquinic acid, derivatives of3,5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives ofaurintricarboxylic acid, caffeic acid phenethyl ester, derivatives ofcaffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin,quercetin, derivatives of quercetin, S-1360, zintevir (AR-177),L-870812, and L-25 870810, MK-0518, BMS-538158, or GSK364735C. See, U.S.Patent Appln. Pub. 2018/0050000.

The patient may be monitored for improvement of the symptoms of ALS.Such symptoms include difficulty walking or doing normal dailyactivities, tripping and falling, weakness of the legs, feet or ankles,hand weakness or clumsiness, slurred speech or trouble swallowing,muscle cramps, twitching in the arms, shoulders or tongue, inappropriatecrying, cognitive changes, and behavior changes.

Any change in symptoms may be monitored for 1-36 months or more, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or36 months. In some embodiments, the decrease of the underlying pathologyis reversed or delayed for at 1-36 months, e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the firstadministration of islatravir.

In some embodiments, the second therapeutic agent is a nucleosidereverse transcriptase inhibitor (NRTI). In some embodiments, the NRTI isabacavir, lamivudine, zidovudine, emtricitabine, tenofovir disoproxilfumarate, tenofovir alafenamide, didanosine, stavudine, apricitabine,alovudine, dexelvucitabine, amdoxovir, fosalvudine or elvucitabine. Inother embodiments, the RTI is abacavir (Ziagen), abacavir/lamivudine(Epzicom), abacavir/lamivudine/zidovudine (Trizivir),lamivudine/zidovudine (Combivir), lamivudine (Epivir), zidovudine(Retrovir), emtricitabine/tenofovir disoproxil fumarate (Truvada),emtricitabine (Emtriva), tenofovir disoproxil fumarate (Viread),emtricitabine/tenofovir alafenamide (Descovy), didanosine (Videx),didanosine extended-release (Videx EC), or stavudine (Zerit). In anotherembodiment, the NRTI is censavudine.

In some embodiments, second therapeutic agent is a non-nucleosidereverse transcriptase inhibitor (NNRTI). In some embodiments, the atleast one NNRTI is Efavirenz (EFV), Nevirapine (NVP), Delavirdine (DLV),Etravirine or Rilpivirine.

In a further embodiment, the second therapeutic agent inhibits L1reverse transcriptase activity in a cell, e.g., a brain cell, of thepatient.

Where the reverse transcriptase inhibitor (RTI) is an FDA approved drug,the RTI may be administered in therapeutically effective amounts thatare approved for therapeutic use. In other embodiments, the amountseffective can be determined with no more than routine experimentation.For example, amounts effective may range from about 1 ng/kg to about 200mg/kg, about 1 μg/kg to about 100 mg/kg, or about 1 mg/kg to about 50mg/kg. The dosage of a composition can be at any dosage including, butnot limited to, about 1 μg/kg. The dosage of a composition may be at anydosage including, but not limited to, about 1 μg/kg, about 10 μg/kg,about 25 μg/kg, about 50 μg/kg, about 75 μg/kg, about 100 μg/kg, about125 μg/kg, about 150 μg/kg, about 175 μg/kg, about 200 μg/kg, about 225μg/kg, about 250 μg/kg, about 275 μg/kg, about 300 μg/kg, about 325μg/kg, about 350 μg/kg, about 375 μg/kg, about 400 μg/kg, about 425μg/kg, about 450 μg/kg, about 475 μg/kg, about 500 μg/kg, about 525μg/kg, about 550 μg/kg, about 575 μg/kg, about 600 μg/kg, about 625μg/kg, about 650 μg/kg, about 675 μg/kg, about 700 μg/kg, about 725μg/kg, about 750 μg/kg, about 775 μg/kg, about 800 μg/kg, about 825μg/kg, about 850 μg/kg, about 875 μg/kg, about 900 μg/kg, about 925μg/kg, about 950 μg/kg, about 975 μg/kg, about 1 mg/kg, about 5 mg/kg,about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg,about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200mg/kg, or more. In other embodiments, the dosage is 1 mg-500 mg. In someembodiments, the dosage is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, or 150 mg. These doses may be unitary or divided and maybe administered one or more times per day. The above dosages areexemplary of the average case, but there can be individual instances inwhich higher or lower dosages are merited, and such are within the scopeof this disclosure. In practice, the physician determinestherapeutically effective amounts and the actual dosing regimen that ismost suitable for an individual subject, which can vary with the age,weight, and response of the particular subject.

The RTI may be administered once, twice or three times per day for 1 dayto the end of life, or for 1 day to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ormore years, or until the RTI causes unacceptable side effects or is nolonger useful.

In some embodiments, islatravir is formulated as part of an implant asdisclosed in US 2019/0388590. In some embodiments, the implant comprisesa biocompatible, nonerodible polymer and islatravir, wherein the implantis implanted sub-dermally and islatravir is continually released in vivoat a rate resulting in a plasma concentration between 0.02 ng/mL and300.0 ng/mL. In some embodiments, islatravir plasma concentration isbetween 0.02 ng/mL and 30.0 ng/mL. In some embodiments, islatravirplasma concentration is between 0.02 ng/mL and 8.0 ng/mL.

In some embodiments, the biocompatible, nonerodible polymer is selectedfrom the group consisting of ethylenevinylacetate copolymer (EVA),poly(urethane), silicone, crosslinked poly(vinyl alcohol),poly(hydroxyethylmethacrylate), acyl substituted cellulose acetates,partially hydrolyzed alkylene-vinyl acetate copolymers, completelyhydrolyzed alkylene-vinyl acetate copolymers, unplasticized polyvinylchloride, crosslinked homopolymers of polyvinylacetate, crosslinkedcopolymers of polyvinyl acetate, crosslinked polyesters of acrylic acid,crosslinked polyesters of methacrylic acid, polyvinyl alkyl ethers,polyvinyl fluoride, polycarbonate, polyamide, polysulphones, styreneacrylonitrile copolymers, crosslinked poly(ethylene oxide),poly(alkylenes), poly(vinyl imidazole),poly(esters), poly(ethyleneterephthalate), polyphosphazenes, chlorosulphonated polylefins, andcombinations thereof. In some embodiments, the biocompatible,nonerodible polymer is ethylene vinyl acetate copolymer.

In some embodiments, the biocompatible, nonerodible polymer is selectedfrom the group consisting ethylene vinylacetate copolymer (9% vinylacetate), ethylene vinyl acetate copolymer (15% vinyl acetate), ethylenevinyl acetate copolymer (28% vinylacetate), and ethylene vinyl acetatecopolymer (33% vinyl acetate). In some embodiments, the biocompatible,nonerodible polymer is ethylene vinyl acetate copolymer (9%vinylacetate). In some embodiments, the biocompatible, nonerodiblepolymer is ethylene vinyl acetate copolymer (15% vinylacetate).

In some embodiments, the biocompatible, nonerodible polymer ispoly(urethane).

In some embodiments, the implant further comprises a diffusional barrierselected from the group consisting of ethylenevinylacetate copolymer(EVA), poly(urethane), silicone, crosslinked poly(vinyl alcohol),poly(hydroxy ethylmethacrylate), acyl substituted cellulose acetates,partially hydrolyzed alkylene-vinyl acetate copolymers, completelyhydrolyzed alkylene-vinyl acetate copolymers, unplasticized polyvinylchloride, crosslinked homopolymers of polyvinylacetate, crosslinkedcopolymers of polyvinyl acetate, crosslinked polyesters of acrylic acid,crosslinked polyesters of methacrylic acid, polyvinyl alkyl ethers,polyvinyl fluoride, polycarbonate, polyamide, polysulphones, styreneacrylonitrile copolymers, crosslinked poly(ethylene oxide),poly(alkylenes), poly(vinyl imidazole), poly(esters),poly(ethyleneterephthalate), polyphosphazenes, chlorosulphonatedpolylefins, and combinations thereof. In some embodiments, thediffusional barrier is ethylene vinyl acetate copolymer. In someembodiments, the diffusional barrier is poly(urethane).

In some embodiments, islatravir is dispersed or dissolved in thebiocompatible nonerodible polymer.

In some embodiments, islatravir is present in the biocompatible,nonerodible polymer between 0.10% to 80% by weight of drug loading. Insome embodiments, islatravir is present at in the biocompatible,nonerodible polymer between 30% to 65% by weight of drug loading. Insome embodiments, islatravir is present in the biocompatible,nonerodible polymer between 40% to 50% by weight of drug loading.

In some embodiments, the implant further comprises between 1% and 20% byweight of a radiopaque material. The radiopaque component will cause theimplant to be X-ray visible. The radiopaque component can be any suchelement known in the art, such as barium sulphate, titanium dioxide,bismuth oxide, tantalum, tungsten or platinum. In a specific embodiment,the radiopaque component is barium sulphate.

In some embodiments, radiopaque material is about 1% to 30% by weight.In another embodiment, the radiopaque material is about 1% to 20% byweight. In another embodiment, the radiopaque material is about 4% to25% by weight. In further embodiment, the radiopaque material is about6% to 20% by weight. In another embodiment, the radiopaque material isabout 4% to 15% by weight. In another embodiment, the radiopaquematerial is about 8% to 15% by weight.

In some embodiments, islatravir is released at therapeuticconcentrations for a duration from between three months and thirty-sixmonths. In some embodiments, islatravir is released at prophylacticconcentrations for a duration from between three months and thirty-sixmonths.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A method for treating, preventing and/or reversing        age-associated inflammation in a patient in need thereof        comprising administering a therapeutically effective amount of a        reverse transcriptase inhibitor (RTI) to the patient in need        thereof, wherein the RTI comprises censavudine or elvucitabine.    -   2. The method of paragraph 1, wherein the age-associated        inflammation is in a patient having Alzheimer's disease,        amyotrophic lateral sclerosis (ALS), Parkinson's disease,        Huntington's disease, vision loss, hearing loss, peripheral        degenerative diseases, or cardiovascular dysfunction,        frontotemporal dementia (FTD), multiple sclerosis (MS), Aicardi        Goutiere's syndrome, progressive supra nuclear palsy (PSP),        osteoarthritis, skin aging, atherosclerosis,        chemotherapy-induced adverse effects, hematopoietic stem cell        function, osteoporosis, physical function, and/or pulmonary        fibrosis, or in a patient in need of wound healing or tissue        regeneration.    -   3. The method of paragraph 1, wherein the age-associated        inflammation is in a patient having Alzheimer's disease.    -   4. The method of paragraph 1, wherein the age-associated        inflammation is in a patient having ALS.    -   5. A method for delaying or reversing the progression of the        underlying pathology of a disorder caused by age-associated        inflammation, comprising administering to a patient in need        thereof a therapeutically effective amount of a reverse        transcriptase inhibitor (RTI), wherein the RTI comprises        censavudine or elvucitabine.    -   6. The method of paragraph 5, wherein the patient has        Alzheimer's disease or ALS and experiences a decrease in one or        more symptoms of Alzheimer's disease or ALS compared to before        the first administration to the patient.    -   7. The method of paragraph 6, wherein the patient has        Alzheimer's disease and the one or more symptoms comprise memory        loss, misplacing items, forgetting the names of places or        objects, repeating questions, being less flexible, confusion,        disorientation, obsessive behavior, compulsive behavior,        delusions, aphasia, disturbed sleep, mood swings, depression,        anxiety, frustration, agitation, difficulty in performing        spatial tasks, agnosia, difficulty with ambulation, weight loss,        loss of speech, loss of short term memory or loss of long term        memory.    -   8. The method of paragraph 6, wherein the patient has        Alzheimer's disease and the decrease in the one or more symptoms        are evaluated according to the DSM-5.    -   9. The method of paragraph 6, wherein the patient has        Alzheimer's disease and the decrease of symptoms is determined        using the cognitive subscale of the Alzheimer's Disease        Assessment Scale (ADAS-cog).    -   10. The method of paragraph 6, wherein the patient has        Alzheimer's disease and the decrease of symptoms is determined        using the Clinician's Interview-Based Impression of Change        (CIBIC-plus).    -   11. The method of paragraph 6, wherein the patient has        Alzheimer's disease and the decrease of symptoms is determined        using the Activities of Daily Living Scale (ADL).    -   12. The method of any one of paragraphs 6-11, wherein the        decrease of symptoms is for 1-36 months.    -   13. The method of any one of paragraphs 6-11, wherein any change        in the underlying pathology is identified by detection of a        biomarker before and after the RTI administration.    -   14. The method of paragraph 13, wherein the biomarker is        β-amyloid or Tau protein.    -   15. The method of paragraphs 13 or 14, wherein the biomarker is        detected by PET imaging.    -   16. The method of paragraphs 13 or 14, wherein the biomarker is        detected by measurement in the cerebrospinal fluid.    -   17. The method of any one of paragraphs 6-11, wherein the        underlying pathology is identified by measurement of brain        volume before and after the RTI administration.    -   18. The method of any one of paragraphs 6-17, wherein the        decrease of the underlying pathology is reversed or delayed for        1-36 months.    -   19. A method for preventing the onset of Alzheimer's disease in        a patient suspected of having mild cognitive impairment,        comprising administering to a patient in need thereof a        therapeutically effective amount of a reverse transcriptase        inhibitor (RTI), wherein the RTI comprises censavudine or        elvucitabine.    -   20. The method of any one of paragraphs 1-19, wherein the        patient has Alzheimer's disease or mild cognitive impairment,        and further comprising administering at least one second        therapeutic agent useful for the treatment of the symptoms of        Alzheimer's disease.    -   21. The method of paragraph 20, wherein the at least one second        therapeutic agent is donepezil, galantamine, rivastigmine, or        memantine.    -   22. The method of paragraph 20, wherein the at least one second        therapeutic agent is an antibody that binds to β-amyloid or Tau        protein.    -   23. The method of paragraph 22, wherein the antibody is binds to        β-amyloid and is bapineuzumab.    -   24. The method of paragraph 22, wherein the antibody binds to        Tau protein and is ABBV-8E12.    -   25. The method of paragraph 20, wherein the at least one second        therapeutic agent is a vaccine against β-amyloid or Tau protein.    -   26. The method of paragraph 20, wherein the at least one second        therapeutic agent is an agent that reduces or alters the brain        content of β-amyloid or Tau.    -   27. The method of paragraph 26, wherein the second therapeutic        agent reduces or alters the brain content of β-amyloid and is a        β-secretase 1 (BACE) inhibitor.    -   28. The method of paragraph 27, wherein the BACE inhibitor is        CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293) or        LY2886721.    -   29. The method of paragraph 26, wherein the second agent reduces        or alters the brain content of Tau and is nicotinamide, or        MPT0G211.    -   30. The method of any one of paragraphs 1, 2, or 4, wherein the        patient has ALS, and further comprising administering at least        one second agent useful for the treatment of ALS.    -   31. The method of paragraph 30, wherein the drug useful for the        treatment of ALS is edaravone or riluzole.    -   32. The method of paragraph 30, wherein the at least one second        agent is an integrase inhibitor.    -   33. The method of paragraph 32, wherein the integrase inhibitor        is raltegravir, curcumin, derivatives of curcumin, chicoric        acid, derivatives of chicoric acid, 3,5-dicaffeoylquinic acid,        derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic        acid, derivatives of aurintricarboxylic acid, caffeic acid        phenethyl ester, derivatives of caffeic acid phenethyl ester,        tyrphostin, derivatives of tyrphostin, quercetin, derivatives of        quercetin, S-1360, zintevir (AR-177), L-870812, and L-25 870810,        MK-0518, BMS-538158, or GSK364735C.    -   34. The method of any one of paragraphs 1-33, wherein the        patient is evaluated for one or more symptoms or disease        pathology 1-36 months after the first administration to the        patient of the RTI.    -   35. The method of any one of paragraphs 1-34, wherein the RTI        inhibits L1 reverse transcriptase activity in a cell of the        patient.    -   36. The method of any one of paragraphs 1-35, wherein the RTI is        elvucitabine.    -   37. The method of any one of paragraphs 1-35, wherein the RTI is        censavudine.    -   38. The method of any one of paragraphs 1-35 further comprising        administering at least one second therapeutic agent to the        patient.    -   39. The method of paragraph 38, wherein the patient has        Alzheimer's disease and the at least one second therapeutic        agent is useful for the treatment of the symptoms of Alzheimer's        disease.    -   40. The method of paragraph 38, wherein the patient has        amyotrophic lateral sclerosis (ALS) and the at least one second        therapeutic agent is useful for the treatment of ALS.    -   41. A method for treating, preventing, delaying the progression        of the underlying pathology, and/or reversing the underlying        pathology of a disease, condition, or disorder in patient in        need thereof, the method comprising administering a        therapeutically effective amount of islatravir, censavudine, or        elvucitabine to the patient, wherein the disease, condition, or        disorder is Alzheimer's disease, amyotrophic lateral sclerosis        (ALS), atherosclerosis, autism spectrum disorder (ADS),        autoimmune disease, cardiovascular dysfunction,        chemotherapy-induced adverse effects, dementia with lewy Bodies        (DLB), frontotemporal dementia (FTD), hearing loss,        hematopoietic stem cell function, Huntington's disease, mild        cognitive impairment (MCI), multi systems atrophy (MSA),        corticobasal degeneration (CDB), multiple sclerosis (MS),        osteoarthritis, osteoporosis, Parkinson's disease, peripheral        degenerative disease, physical function, progressive supra        nuclear palsy (PSP), pulmonary fibrosis, Rett Syndrome,        schizophrenia, skin aging, vision loss, or in a patient in need        of wound healing or tissue regeneration, or in a patient in need        of wound healing or tissue regeneration.    -   42. The method of paragraph 41 for treating or preventing the        disease, condition, or disorder in the subject in need thereof.    -   43. The method of paragraph 41 for delaying the progression of        the underlying pathology or reversing the underlying pathology        of the disease, condition, or disorder in the subject in need        thereof.    -   44. The method of any one of paragraphs 41-43, wherein the        disease, condition, or disorder is Alzheimer's disease, ALS,        FTD, PSP, or Rett Syndrome.    -   45. The method of paragraph 44, wherein the disease, condition,        or disorder is Alzheimer's disease or ALS, and the patient        experiences a decrease in one or more symptoms of Alzheimer's        disease or ALS after administration of islatravir, censavudine,        or elvucitabine to the patient.    -   46. The method of paragraph 45, wherein the disease, condition,        or disorder is Alzheimer's disease, and the one or more symptoms        comprise memory loss, misplacing items, forgetting the names of        places or objects, repeating questions, being less flexible,        confusion, disorientation, obsessive behavior, compulsive        behavior, delusions, aphasia, disturbed sleep, mood swings,        depression, anxiety, frustration, agitation, difficulty in        performing spatial tasks, agnosia, difficulty with ambulation,        weight loss, loss of speech, loss of short term memory or loss        of long term memory.    -   47. The method of paragraph 45, wherein the disease, condition,        or disorder is Alzheimer's disease, and the decrease in the one        or more symptoms are evaluated according to the DSM-5.    -   48. The method of paragraph 45, wherein the disease, condition,        or disorder is Alzheimer's disease, and the decrease of symptoms        is determined using the cognitive subscale of the Alzheimer's        disease Assessment Scale (ADAS-cog).    -   49. The method of paragraph 45, wherein disease, condition, or        disorder is Alzheimer's disease, and the decrease of symptoms is        determined using the Clinician's Interview-Based Impression of        Change (CIBIC-plus).    -   50. The method of paragraph 45, wherein the disease, condition,        or disorder is Alzheimer's disease and the decrease of symptoms        is determined using the Activities of Daily Living Scale (ADL).    -   51. The method of any one of paragraphs 45-50, wherein the        decrease of symptoms is for 1-36 months.    -   52. The method of paragraph 43, wherein the underlying pathology        is identified by detection of a biomarker before and after        administration of islatravir, censavudine, or elvucitabine to        the patient.    -   53. The method of paragraph 52, wherein the biomarker is        β-amyloid or Tau protein.    -   54. The method of paragraph 52 or 53, wherein the biomarker is        detected by PET imaging.    -   55. The method of paragraph 52 or 53, wherein the biomarker is        detected by measurement in the cerebrospinal fluid.    -   56. The method of any one of paragraphs 52-55, wherein the        underlying pathology is identified by measurement of brain        volume before and after islatravir, censavudine, or elvucitabine        administration.    -   57. The method of paragraph 43, the underlying pathology is        delayed or reversed for 1-36 months.    -   58. The method of paragraph 41, wherein disease, condition, or        disorder is an autoimmune disease.    -   59. The method of paragraph 58, wherein the autoimmune disease        is Aicardi Goutiere's Syndrome (AGS), lupus, or rheumatoid        arthritis.    -   60. The method of any one of paragraph 41-59, further comprising        administering a therapeutically effective amount of at least one        second therapeutic agent useful for treating or preventing the        disease, condition, or disorder.    -   61. The method of paragraph 60, wherein the at least one second        therapeutic agent is a nucleoside reverse transcriptase        inhibitor (NRTI).    -   62. The method of paragraph 61, wherein the at least one NRTI is        abacavir (Ziagen), abacavir/lamivudine (Epzicom),        abacavir/lamivudine/zidovudine (Trizivir), lamivudine/zidovudine        (Combivir), lamivudine (Epivir), zidovudine (Retrovir),        emtricitabine/tenofovir disoproxil fumarate (Truvada),        emtricitabine (Emtriva), tenofovir disoproxil fumarate (Viread),        emtricitabine/tenofovir alafenamide (Descovy), didanosine        (Videx), didanosine extended-release (Videx EC), stavudine        (Zerit), apricitabine, alovudine, dexelvucitabine, amdoxovir,        fosalvudine or elvucitabine.    -   63. The method of paragraph 60, wherein the at least one second        therapeutic agent is a non-nucleoside reverse transcriptase        inhibitor (NNRTI).    -   64. The method of paragraph 63, wherein the NNRTI is Efavirenz        (EFV), Nevirapine (NVP), Delavirdine (DLV), Etravirine or        Rilpivirine.    -   65. The method of paragraph 60, wherein the disease, condition,        or disorder is Alzheimer's disease or mild cognitive impairment,        and the at least one second therapeutic agent is useful for the        treatment of the symptoms of Alzheimer's disease.    -   66. The method of paragraph 65, wherein the at least one second        therapeutic agent is donepezil, galantamine, rivastigmine, or        memantine.    -   67. The method of paragraph 65, wherein the at least one second        therapeutic agent is an antibody that binds to β-amyloid or Tau        protein.    -   68. The method of paragraph 67, wherein the antibody binds to        β-amyloid and is bapineuzumab.    -   69. The method of paragraph 67, wherein the antibody binds to        Tau protein and is ABBV-8E12. 70. The method of paragraph 60,        wherein the at least one second therapeutic agent is a vaccine        against β-amyloid or Tau protein.    -   71. The method of paragraph 60, wherein the at least one second        therapeutic agent is an agent that reduces or alters the brain        content of β-amyloid or Tau.    -   72. The method of paragraph 71 wherein the second therapeutic        agent reduces or alters the brain content of β-amyloid is a        β-secretase 1 (BACE) inhibitor.    -   73. The method of paragraph 72, wherein the BACE inhibitor is        CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293) or        LY2886721.    -   74. The method of paragraph 71, wherein the second agent reduces        or alters the brain content of Tau is nicotinamide, or MPT0G211.    -   75. The method of paragraph 60, wherein the patient has ALS, and        further comprises administering at least one second therapeutic        agent useful for the treatment of ALS.    -   76. The method of paragraph 75, wherein the second therapeutic        agent is edaravone or riluzole.    -   77. The method of paragraph 75, wherein the at least one second        therapeutic agent is an integrase inhibitor.    -   78. The method of paragraph 77, wherein the integrase inhibitor        is raltegravir, curcumin, derivatives of curcumin, chicoric        acid, derivatives of chicoric acid, 3,5-dicaffeoylquinic acid,        derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic        acid, derivatives of aurintricarboxylic acid, caffeic acid        phenethyl ester, derivatives of caffeic acid phenethyl ester,        tyrphostin, derivatives of tyrphostin, quercetin, derivatives of        quercetin, S-1360, zintevir (AR-177), L-870812, and L-25 870810,        MK-0518, BMS-538158, or GSK364735C.    -   79. The method of any one of paragraphs 41-78, wherein the        patient is evaluated for one or more symptoms or disease        pathology 1-36 months after the first administration to the        patient of islatravir, censavudine, or elvucitabine.    -   80. The method of any one of paragraphs 41-79, wherein the        patient is (a) not infected with the HIV virus, (b) is not        suspected of being infected with the HIV virus, and/or (c) is        not being treated to prevent infection with the HIV virus.    -   81. The method of any one of paragraphs 41-80 comprising        administering a therapeutically effective amount of islatravir        to the patient in need thereof.    -   82. The method of paragraph 81, wherein islatravir is        administered daily.    -   83. The method of paragraph 82, wherein islatravir is        administered daily in an amount about 0.1 mg to about 10 mg.    -   84. The method of paragraph 81, wherein islatravir is        administered monthly.    -   85. The method of paragraph 84, wherein islatravir is        administered monthly in an amounts of about 60 or about 120 mg.    -   86. The method of paragraph 81, wherein islatravir is        administered from an implant that contains 54 or 62 mg of        islatravir.    -   87. The method of any one of paragraphs 41-80 comprising        administering a therapeutically effective amount of censavudine        to the patient in need thereof.    -   88. The method of any one of paragraphs 41-80 comprising        administering a therapeutically effective amount of elvucitabine        to the patient in need thereof.

Examples

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention and are not intended to limit the invention.

Methods

Cell Culture

Several different strains of normal human fibroblasts were employed inthis study. LF1 cells were derived from embryonic lung tissue asdescribed.⁵⁸ These cells have been in continuous use in our laboratorysince their isolation in 1996. For this study original samples frozen in1996 and in continuous storage in our laboratory were recovered andused. IMR-90 and WI-38 cells were obtained from the ATCC. None of thesecell lines are listed in the International Cell Line AuthenticationCommittee (ICLAC) database. These normal fibroblast cell lines werecultured using physiological oxygen conditions (92.5% N₂, 5% CO₂, 2.5%02), in Ham's F-10 nutrient mixture (Thermo Scientific) with 15% fetalbovine serum (FBS, Hyclone). Medium was additionally supplemented withL-glutamine (2 mM), penicillin and streptomycin.⁵⁹ Cell cultures wereperiodically tested for mycoplasma contamination with MycoAlert®Mycoplasma Detection Kit (Lonza).

To obtain replicatively senescent (RS) cells, LF1 cultures were seriallypropagated until proliferation ceased. At each passage, after reaching80% confluence cells were trypsinized and diluted 1:4. Hence eachpassage is equivalent to approximately two population doublings. Inearly passage cultures, the time between passages is constant atapproximately 3 days. As cultures approached senescence the time betweenpassages gradually increased. An interval of 2-3 weeks indicated thatthe culture was in its penultimate passage. At this point, afterreaching 80% confluence, the cells were replated at 1:2 dilution, andthis time was designated as the last passage (point A in FIG. 2A). Somecell growth typically does occur in the next 2-3 weeks, but the culturesdo not reach 80%. Under this experimental regimen, the majority of thecells in the culture enter senescence within a 3-4 weeks window centeredroughly around the time of last passage (grey bar in FIG. 2A). At pointB (4 weeks), the cultures were trypsinized and replated as described⁶⁰to eliminate a small fraction of persisting contact-inhibited cells.Cultures were again replated at point C (8 weeks).

Oncogene-induced senescence (OIS) was elicited by infectingproliferating LF1 cells with pLenti CMV RasV12 Neo (Addgene plasmid#22259). Generation of lentiviral particles and the infection procedureare described below. At the end of the infection, cells were reseeded at15-20% confluency and selected with G418 (250 μg/ml) maintainedcontinuously until the end of the experiment Medium was changed every 3days until the cultures were harvested at the indicated time points.Stress-induced premature senescence (SIPS) was elicited by X-rayirradiation with 20 Gy given at a rate of 87 cGy/min in one fractionusing a cesium-137 gamma source (Nordion Gammacell 40). Cells were15-20% confluent at the time of irradiation. Medium was changedimmediately after irradiation, and at 3-day intervals thereafter. 293Tcells (Clontech) were used to package lentivirus vectors and werecultured at 37° C. in DMEM with 10% FBS under normoxic conditions (airsupplemented with 5% CO₂).

Reverse Transciptase Inhibitors (RTIs)

All RTIs (lamivudine, 3TC; zidovudine, AZT; abacavir, ABC;emtricitabine, FTC) used in this study were USP grade and obtained fromAurobindo Pharma, Hyderabad, India. For Trizivir (TZV), its constituents(ABC, AZT and 3TC) were combined in the appropriate amounts.

Mouse Husbandry

C57BL6J mice of both sexes were obtained from the NIA Aged RodentColonies⁶¹ at 5 and 18 months of age. The 5-month old animals weresacrificed after a short (1 week) acclimatization period, a variety oftissues were harvested, snap frozen in LN2 and stored at −80° C. The18-month old animals were housed until they reached a desired age. Micewere housed in a specific pathogen-free AAALAC-certified barrierfacility. Cages, bedding (Sani-chip hardwood bedding) and food (PurinaLab Chow 5010) were sterilized by autoclaving. Food and water (alsosterilized) were provided ad libitum. A light-dark cycle of 12 hours wasused (7 AM On, 7 PM Off). Temperature was maintained at 70° F., andhumidity at 50%. All animals were observed daily and weighed once perweek. In a pilot experiment, three cohorts of 10 animals each weretreated with 3TC dissolved in drinking water (1.5 mg/ml, 2.0 mg/ml, 2.5mg/ml) continuously from 18 months until sacrifice at 24 months. Thefourth (control) cohort was provided with the same water without drug.No significant differences in behavior, weight, or survival wereobserved between the 4 cohorts during the entire experiment. Once duringthe experiment (at 20 months of age) the animals were subjected to asingle tail bleed of approximately 70 μL. The collected plasma wasshipped to the University of North Carolina CFAR Clinical Pharmacologyand Analytical Chemistry Core for analysis of 3TC. For the 2 mg/mLcohort the concentration of 3TC in plasma averaged 7.2 μM. This dose ofdrug was chosen for further experiments to mimic the human HIVtherapeutic dose (300 mg per day, 5-8 μM in plasma).⁶² For theexperiments presented in this communication animals were aged in houseuntil they reached 26 months of age. They were then assigned randomly totwo cohorts by a technician that was blinded to the appearance or othercharacteristics of the animals. One cohort was treated for 2 weeks with2 mg/mL of 3TC in drinking water, and the other (control) cohort withsame water without drug, administered in the same manner. At the end ofthe treatment period all animals were sacrificed and harvested fortissues as described above. All the animals in both cohorts were allincluded in all subsequent analyses. The experiment was performed onseparate occasions with male and female animals. Non-lethal total bodyirradiation (6 Gy) was performed as described⁶³ and tissue specimenswere kept on dry ice.

PCR

The ABI ViiA 7 instrument (Applied Biosystems) was used for allexperiments. qPCR of DNA was performed using the TaqMan system (AppliedBiosystems) as described by Coufal et al. (2009).⁶⁴ 100 μg of purifiedgenomic DNA was used with the indicated primers (see Table 1). Reversetranscription qPCR (RT-qPCR) of RNA was performed using the SYBR Greensystem (Applied Biosystems). Polyadenylated RNA was used in allexperiments assessing transcription of L1 elements, and total RNA wasused for all other genes. Total RNA was harvested using the Trizolreagent (Invitrogen). Poly(A) RNA was isolated from total RNA using theNEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). 1μg of total RNA, or 10 ng of poly(A) RNA, were reverse-transcribed intocDNA in 50 μL reactions using the TaqMan kit (Applied Biosystems). Toassess strand-specific transcription, the random primers in the RTreaction were substituted with a strand-specific primer to the targetRNA. 1 μL of each RT reaction was used in subsequent qPCR reactions.GAPDH was used as the normalization control in experiments with humancells. The arithmetic mean of Gapdh and two additional controls (Hsp90and GusB) was used for normalization of RT-qPCR experiments with murinetissues, with the exception of liver that was normalized to Hsp90 andGusB. For measuring L1 transcription, poly(A) RNA samples wereexhaustively digested with RNase-free DNase (Qiagen) prior to thesynthesis of cDNA6. Effectiveness of the DNase digestion was assessedusing controls that omitted the RT enzyme.

Design of PCR Primers

Primer sets 1 to 5 (Table 1, amplicons A to E in FIG. 1B) to human L1were designed to preferentially amplify elements of the human-specificL1HS and evolutionarily recent primate-specific L1PA(2-6) subfamilies,as follows. First, the consensus sequences of L1HS and L1PA2 throughL1PA6 elements were obtained from Repbase (Genetic Information ResearchInstitute⁶⁵). Second, a consensus sequence of these six sequences wasgenerated with the Clustal Omega multiple sequence alignmenttool.^(66,67) Primer design was then done on the overall consensus withPrimer3 and BLAST using the NCBI Primer-BLAST tool.^(68,69) L1 primerpairs were evaluated for their targets using the In-Silico PCR⁷⁰ toolagainst the latest genome assembly (hg38) with a minimum perfect matchon 3′ end of each primer equal to 15. Primers to ORF2 (primer set 6,amplicon F in FIG. 1B) were developed by Coufal et al. (2009) topreferentially target L1HS. Primers to assess transcription of activemurine L1 elements (primer set 37, Table 1) were designed on thecombined consensus sequence of the L1MdA and L1Tf families obtained fromRepbase and validated as described above. L1 primer pairs, spanning thefull length these elements (primer sets 48-50), were designed using thesame strategy. Primer pairs specific to the three active families ofmurine L1 elements (primer sets 51-53) were designed exploitingpolymorphisms in the 5′UTR region. RT-qPCR analysis of L1 transcriptionwas performed on poly(A) purified RNA using the SYBR Green method. Forall other (non-1) genes, whenever possible, primers are separated by atleast one intron in the genomic DNA sequence (as indicated in Table 1).Primers to the human IFN alpha family were designed against a consensussequence of all the human IFN alpha gene sequences (IFNA1, IFNA2, IFNA4,IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17,IFNA21) generated with the Clustal Omega multiple sequence alignmenttool. All primers against murine targets were designed as describedabove and are listed in Table 1. Sequences of primers corresponding to aconsensus of all the murine IFN alpha family genes, as well as to theIFNB1 gene, were obtained from the publication by Gautier et al.⁷¹ Forquantifying relative L1 genomic copy number (human cells), the TaqManmultiplex method developed by Coufal et al. (2009)⁷² was used. Theseprimers are listed as set #6 and set #7 (with their corresponding VICand 6FAM probes) in Table 1.

Chromatin Immunoprecipitation

All ChIP experiments were performed using the Chromatrap spin columnChIP kit (Porvair). Briefly, 2×10⁶ cells were crosslinked in theirculture dishes with 1% formaldehyde (10 min, room temperature), quenchedwith glycine, washed twice with ice-cold PBS (containing proteaseinhibitors), and finally scraped into a microfuge tube. Cell pelletswere resuspended in 0.4 mL of hypotonic buffer and incubated for 10 minon ice. Nuclei were spun down, resuspended in 0.3 mL lysis buffer, andsonicated using a Bioruptor UCD-200 instrument (Diagenode) set to pulseon high (30 sec followed by 30 sec. rest) for a total time of 10 min Theextracts were centrifuged in a microfuge (top speed, 5 min, 4° C.) toremove debris, the supernatants were transferred to new tubes, andstored at −80° C. An amount of extract containing 2 μg of DNA wascombined with 4 μg of antibody and loaded on a Chromatrap solid phaseProtein A matrix. Immunocomplexes were allowed to form overnight at 4°C. with mild agitation, following which the samples were washed andeluted according to the manufacturers protocol. Rabbit IgG and 1% inputwere used as controls. 1 μL of immunoprecipitated DNA was used in eachqPCR reaction.

BrdU Pull-Down

To obtain quiescent cells, proliferating cells were grown to 50%confluence, serum supplementation of the medium was changed to 0.1% FBS,and incubation was continued until harvest. Quiescent and senescentcells were continuously labeled for two weeks with BrdU (BrdU LabelingReagent, Thermo Fisher) according to the manufacturer's protocol forlabeling of culture cells. Cell were harvested and counted: 5×10⁵ cellswere processed per condition. Genomic DNA was purified viaPhenol:Chloroform extraction, RNase A treated and subsequently shearedusing a Bioruptor UCD-200 instrument (pulse on Low, 30 sec on and 30 secoff, 10 min total). DNA tubes were incubated in a heat block (100° C.)for exactly one minute and then flash frozen in liquid nitrogen. Tubeswere let thaw at room temperature and 1 μg of purified anti-BrdUantibody (BD Pharmingen, Cat. #555627) was added per tube together withmagnetic protein A/G beads and ChIP Dilution Buffer. Immuno-slurrieswere incubated overnight at 4° C. with constant rotation.Immuno-captured BrdU labeled DNA was purified according to the MagnaChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore Sigma). UnboundDNA was kept as input. 1 μL of immunoprecipitated DNA was used in eachqPCR reaction. Alternatively, to enrich for single-stranded BrdU-labeledDNA the heat-mediated denaturation was omitted and samples wereprocessed for BrdU pull-down as above. The DNA second strand was thengenerated by adding a mixture of random primers (Thermo Fisher), secondstrand synthesis reaction buffer, dNTPs and DNA Pol I (New EnglandBiolabs). The reaction was incubated for 4 hrs at 16° C. andsubsequently purified by phenol-chloroform extraction. Following thesecond strand synthesis, the dsDNA was end-repaired with the End-It DNAEnd-Repair Kit (Epicenter, Cat. #ER0720). Blunt-ended fragments werecloned using the Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher), andthen used to transform One Shot TOP10 chemically competent E. coli(Thermo Fisher, Cat. #C404010). Individual colonies were picked andsubjected to Sanger sequencing using a T7 promoter primer at BeckmanCoulter Genomics.

RNA-Seq

Total RNA from early passage, early and deep senescent cells (FIG. 6A)was extracted as described above. The total RNA was processed with theIllumina TruSeq Stranded Total RNA Ribo-Zero kit and subjected toIllumina HiSeq2500 2×125 bp paired end sequencing using v4 chemistry atBeckman Coulter Genomics Inc. Over 70 million reads were obtained foreach sample. The RNA-seq experiment was performed in three biologicalreplicates.

Raw RNA-sequencing reads were aligned to the GrCh38 build of the humangenome using HiSat⁷³ Counts mapping to the genome were determined usingfeatureCounts.⁷⁴ Counts were then normalized using the trimmed mean ofM-values (TMM) method in EdgeR.⁷⁵ EdgeR was additionally used to derivedifferential expression from the normalized data set. Differentialexpression data were then ranked by log 2 fold change and input into theGenePattern interface for GSEA Preranked, using 1000 permutations, todetermine enrichment for KEGG pathways, SASP, and the interferonresponse.^(76,77) The outputs were then corrected for multiplecomparisons by adjusting the nominal p value using Benjamini-Hochbergmethod.⁷⁸ Data were displayed using GENE-E software.⁷⁹

In Silico Analysis of Transcription Factors Binding to L1

Transcription factor profiles were created using ChIP-seq data from theENCODE project (GEO accession numbers GSE2961 and GSE32465).Transcription factor ChIP-seq and input control reads were aligned tothe consensus sequence of L1HS using bowtie1.⁸⁰ The log₂ fold changeenrichment was calculated per base pair of the L1HS consensus using thetranscription factor ChIP-seq read coverage per million mapping reads(RPM) versus input control RPM values and smoothed by LOESS smoothingwith a parameter α=0.1. The total number of mapping reads used in RPMnormalization was determined from a separate bowtie1 alignment to thehuman genome (hg19).

Construction of FOXA1 Reporters

L1 promoter reporter plasmids L1WT and L1 del (390-526) were obtainedfrom Sergey Dmitriev, Institute of Bioorganic Chemistry, Moscow.^(81,82)Both contain luciferase as the reporter cloned in the sense orientation.To determine antisense transcription from the same plasmid, EYFP wasinserted in the inverse orientation upstream of the L1 5′ UTR asfollows. The EYFP sequence was excised from pEYFP-N1 (Clontech, Cat.#6006-1) with AgeI and NotI and blunt ended. Plasmids L1WT and L1delwere digested with XbaI, blunted and treated with FastAP (Fermentas).Successful insertion of anti-sense EYFP was verified using PCR primersAAAGTTTCTTATGGCCGGGC (in EYFP) and GCTGAACTTGTGGCCGTTTA (in L1 promoter)and Sanger sequencing. Plasmid pcDNA3.1/LacZ was used as theco-transfection control. Luciferase and β-galactosidase assays wereperformed as described.⁸³ EYFP-N1 was used as a positive control fordetecting the EYFP signal. Co-transfections were performed on earlypassage LF1 cells using Lipofectamine with Plus Reagent (Invitrogen)according to the manufacturers instructions.

Lentiviral Vectors

Constructs were obtained from public depositories as indicated below.Virions were produced and target cells were infected as described.⁸⁴shRNA sequences were obtained from The RNAi Consortium (TRC),⁸⁵ clonedinto third generation pLKO.1 vectors and tested for efficacy. Fourselectable markers were used to allow multiple drug selections: pLKO.1puro (2 μg/ml) and pLKO.1 hygro (200 μg/ml) (Addgene plasmid #8453,24150), pLKO.1 blast (5 μg/ml) (Addgene plasmid #26655), pLKO.1 neo (250μg/ml) (Addgene plasmid 13425). pLK-RB1-shRNA63 and pLK-RB1-shRNA19(Addgene plasmids #25641 and 25640).⁸⁶ For FOXA1 shRNAs TRCN0000014881(a) and TRCN0000014882 (b) were used. For TREX1 shRNAs TRCN0000007902(a) and TRCN0000011206 (b) were used. For knockdown of L1, nine shRNAswere designed and tested, of which two (shL1_11 to ORF1,AAGACACATGCACACGTATGT, and shL1_44 to ORF2 AAGACACATGCACACGTATGT) showedsignificant knockdown (FIG. 10G) and were chosen for further work. Theremaining seven shRNAs produced no or minimal knockdown. For cGAS shRNAsTRCN0000128706 (a) and TRCN0000128310 (b) were used. For STING shRNAsTRCN0000161345 (a) and TRCN0000135555 (b) were used.

All ectopic expression experiments used constructs generated by theORFeome Collaboration⁸⁷ in the lentivirus vector pLX304 (blasticidinresistant, Addgene plasmid 25890) and were obtained from the DNASUplasmid repository.⁸⁸ RB1 (ccsbBroad304_06846, HsCD00434323), TREX1(ccsbBroad304_02667, HsCD00445909), FOXA1 (ccsbBroad304 06385,HsCD00441689).

All the interventions in senescent cells were initiated by infectingcells at 12 weeks of senescence (point D in FIG. 6A). Followingappropriate drug selections, cells were incubated until 16 weeks ofsenescence (point E in FIG. 6A), when they were harvested for furtheranalysis.

The 3× intervention was performed by infecting early passage LF1 cellssequentially with vectors pLKO.1 puro shRB, pLKO.1 hygro shTREX1 andpLX304 blast FOXA1 (FIG. 10C). After each infection, the arising drugresistant pool of cells was immediately infected with the next vector.After the third infection, the cells were harvested for further analysis48 hours after the drug selection was complete. The infections were alsoperformed in various combinations and in each case resulted in theactivation of L1 expression, entry into senescence, and induction of anIFN-I response. The sequence above was chosen because it gave the mostefficient selection of cells for further analysis. To allow anadditional (fourth) intervention in 3× cells (shL1, shSTING, or shCGAS),hairpins targeting RB1 were re-cloned in pLKO.1 neo, thus freeing pLKO.1puro for the fourth gene of interest. This allowed an efficient drugselection process and sample harvest 48 hrs after the last selection.

Retrotransposition Reporters

The two-vector dual luciferase reporter system reported by Xie et al.(2011)⁸⁹ was adapted for lentiviral delivery. The L1RP-Fluc reporterswere recloned from plasmids pWA355 and pWA366 into the lentiviralbackbone pLX304. pWA355 contained a functional, active L1_(RP) element,whereas pWA366 contains L1RP(JM111), a mutated element carrying twomissense mutations in ORF1 that is unable to retrotranspose. Earlypassage LF1 cells were infected with a puromycin resistant lentivirusexpressing Rluc. Pooled drug resistant cells were then infected withhigh titer particles of pLX304-WA355 or pLX304-WA366 constructs.Immediately after infection cells were treated for four days with 3TC(at the indicated concentrations). Cells were then harvested and assayedfor Rluc and Fluc luciferase activities. The native L1retrotransposition reporter pLD143⁹⁰ was co-transfected with pLKOvectors (shLuc, shL1_11 and shL1_44) into HeLa cells using FuGene HD(Promega). Cell culture, transfection and retrotransposition assays weredone as described above. Retrotransposition activity was normalized tothe activity of L1_(RP) co-transfected with shLuc. Three independentexperiments were performed for each construct.

Identification of Expressed L1 Elements by Long-Range RT-PCR and 5′RACE

Total RNA was harvested from cells using the Trizol reagent(Invitrogen). The RNA was further purified using the Purelink RNA Minikit (Invitrogen) with DNase I digestion. From the eluted total RNA,poly(A) RNA was isolated using the NEBNext Poly(A) mRNA MagneticIsolation Module (New England Biolabs). The forward primer(MDL15UTRPRAF, primer set 1, Table 1) was used with either of tworeverse primers (MDL15UTRPRCR, primer set 3, amplicon size 537 bp) orMDL15UTRPRDR, primer set 4, amplicon size 654 bp). A high-fidelitythermostable reverse transcriptase (PyroScript RT-PCR Master Mix Kit,Lucigen) was used with 10 ng of poly(A) mRNA per reaction and amplifiedfor 10 cycles. No template and RNaseA-treated samples were used asnegative controls. The generated amplicons were cloned into the TOPO-TA(Invitrogen) vector and the resulting plasmids were used to transformOne Shot TOP10 chemically competent E. coli. Individual colonies werepicked and subjected to Sanger sequencing using a T7 promoter sequencingprimer at Beckman Coulter Genomics. 96 sequencing reactions (1 plate)were performed for each primer pair in four experiments for a total of768 sequenced clones. Sequencing data were trimmed to remove the RT-PCRprimers and BLASTed against the human genome (GRCh38) with amatch/mismatch cost of +1, −4 and allowing species-specific repeats forHomo sapiens. Only perfect hits were scored and annotated for genomiccoordinates. 658 clones could be mapped to the reference genome, 51contained at least 1 mismatch and thus likely represent elements thatare polymorphic in the cell line, and 58 were cloning artifacts.Whenever a clone presented multiple instances of perfect identity afractional count was adopted, dividing the counts by the number ofelements sharing the same sequence. Each mappable clone was furtheranalyzed using L1Xplorer⁹¹ to recover the classification features of theL1 element and whether it is intact.

Alternatively, poly(A) RNA isolated as above was subjected to RapidAmplification of cDNA Ends (RACE). Each reaction contained 10 ng ofpoly(A) RNA and was processed using the 5′RACE System kit (ThermoFisher, Cat. #18374-041). The two antisense gene-specific primers (GSP)used for 5′RACE were: for GSP1, MDL15UTRPRDR (primer set 4, Table 1),and for the nested GSP2, MDL15UTRPRCR (primer set 3, Table 1).Amplification products were cloned and sequenced as above, using a T7promoter sequencing primer by Beckman Coulter Genomics. A total of 94clones were sequenced; 26 contained mostly a polyG stretch generated bythe tailing step in the RACE protocol and 18 could not be mapped to thehuman genome. The remaining 50 mappable clones contained L1 sequencesand were aligned to the L1HS consensus using a setting of >95% identityat positions 1-450.⁹² The mappable clones were also assigned toindividual L1 families using RepEnrich software.⁹³ Pairwise alignmentsto the consensus were performed with LALIGN.⁹⁴ Multiple sequencealignments were calculated using MAFFT (Multiple Alignment using FastFourier Transform) with the L-INS-i algorithm (accurate for alignmentsof <200 sequences).⁹⁵ Alignment visualization, %-identity coloring andconsensus were generated by Jalview.⁹⁶

Generation and Analysis of CRISPR-Cas9 Knockouts

Three distinct gRNA sequences for each chain of the IFNAR receptor(IFNAR1 and IFNAR2), listed in the GeCKO v2.0 resource (Feng Zhang Lab,MIT⁹⁷)⁹⁸, were tested and the following ones were chosen: IFNAR1(HGUbA_29983) AACAGGAGCGATGAGTCTGTA; IFNAR2 (HGLibA_29985)GTGTATATCAGCCTCGTGTT. Cas9 and gRNAs were delivered using a singlelentivirus vector (LentiCRISPR_v2, Feng Zhang Lab, MIT; Addgene plasmid#52961), carrying a puromycin resistance gene. The efficacy of theCRISPR-Cas9 mutagenesis, on the basis of which the above two gRNAs werechosen, was evaluated by treating the infected and drug-selected cellswith interferon (universal type I interferon, PBL Assay Science, Cat.#11200-1) and monitoring nuclear translocation of phospho-STAT2 and IRF9by immunofluorescence. The absence of translocation signifies lack ofIFN-I responsiveness and hence loss of IFNAR function. Experimentalprocedures followed the protocols provided by the Zhang lab.^(99,100) Inthe experiment shown in FIG. 3H (RS) and FIG. 10K, both IFNAR1 andIFNAR2 gRNAs were used to treat the same cells to further increase theefficacy of ablating the INF-I response. For early passage and senescentcells, co-infections of IFNAR1 and IFNAR2 vectors were performedfollowed by selection with puromycin. For senescent cells, high titerlentivirus particles were applied to senescent cells at 12 weeks insenescence (point D, FIG. 6A) and cells were assayed 4 weeks later(point E, FIG. 6A). For the experiment shown in FIG. 3H (SIPS), editedearly passage cells were single-cell cloned. 24 single cells wereisolated using the CellRaft technology (Cell Microsystems) and expanded.Genomic screening of the CRISPR cut site was performed by the CRISPRSequencing Service (CCIB DNA Core, Massachusetts General Hospital).¹⁰¹The successful knock out of IFNAR1 and IFNAR2 was verified in four outof the 24 expanded clonal cell lines.

Immunoblotting

Cells were harvested in Laemmli sample buffer (60 mM Tris pH 6.8, 2%SDS, 10% glycerol, 100 mM DTT) and boiled for 5 min at 100° C. Wholecell extracts (60 μg protein) were separated by SDS-PAGE and transferredonto Immobilon-FL membranes (Millipore). Nonspecific binding was blockedby incubation in 4% bovine serum albumin (BSA; Thermo Fisher) and 0.1%Tween-20 in PBS for 1 hr at room temperature. Primary antibodies werediluted in the blocking solution and incubated overnight at 4° C. A listof all the primary antibodies is provided in Table 2. Secondaryantibodies were diluted in blocking solution and incubated for 1 hour atroom temperature. Signals were detected using the LI-COR Odysseyinfrared imaging system (LI-COR Biosciences). For the quantification ofsignals all samples to be compared were run on the same gel. Loadingstandards were visualized on the same blot as the test samples using theLI-COR 2-color system. Bands were imaged and quantified using LI-CORsoftware. All bands to be compared were quantified on the same image andwere within the linear range of detection of the instrument.

Immunofluorescence Microscopy Performed on Cells in Culture

Cells were grown on glass cover slips and the samples were processed aspreviously described.¹⁰² Primary antibodies are listed in Table 2.Staining of ssDNA was performed as described by Thomas et al.¹⁰³Briefly, cells seeded on coverslips were fixed on ice with 4%paraformaldehyde (PFA) for 20 min and then incubated in 100% methanol at−20° C. overnight. The cells were then treated with 200 mg/mL RNase A at37° C. for 4 hrs. Cells were blocked with 3% BSA and incubated overnightat 4° C. with primary antibodies diluted in 3% BSA. Images were acquiredusing a Zeiss LSM 710 confocal laser scanning microscope or a Nikon Ti—Sinverted fluorescence microscope. All microscope settings were set tocollect images below saturation and were kept constant for all imagestaken in one experiment, as previously described.¹⁰⁴ Image analysis wasperformed as described below for tissues.

PCR Arrays

Total RNA was harvested from cells as indicated above (Quantitative PCR)and analyzed using the Qiagen RT Profiler™ Human Type I InterferonResponse PCR Array (Cat. #PAHS-016ZE-4). Reverse transcription reactionswere performed with the Qiagen RT² First Strand Kit (Cat. #330404) using1 μg of total RNA as starting material. 102 μL of the completed reactionwas combined with 650 μL of Qiagen RT SYBR Green ROX qPCR Mastermix(Cat. #330521) and 548 μL of RNase-free molecular grade water and run inthe 384-well block on a ViiA 7 Applied Biosystems instrument. Allprocedures followed the manufacturer's protocols. All conditions wererun in triplicate. The results were analyzed using the Qiagen GeneGlobeData Analysis Center.¹⁰⁵ Briefly, C_(t) values were normalized to apanel of housekeeping genes (HKG). ΔC_(t) values ware calculated betweena gene of interest (GOI) and the mean HKG value. Fold changes were thencalculated using 2^(−ΔΔCT) formula. The lower limit of detection was setat C_(t) of 35. For any GOI to be considered significant, the followingfilters were set: (i) >2-fold change in expression; and (ii)p-value >0.05. In addition, genes with an average C_(t)>32 in bothcontrol and test samples were also eliminated.

Enzyme Linked immunosorbent Assay (ELISA)

Interferon β levels were quantified with the VeriKine-HS Human IFN BetaSerum ELISA Kit (PBL Assay Science, Cat. #41415). Cell culture mediawere conditioned for 48 hrs before harvest. To remove particles anddebris, 1 mL aliquots were spun 5 min 5,000×g. All incubations wereperformed in a closed chamber at room temperature (22-25° C.) keepingthe plate away from temperature fluctuations. 50 μL of sample bufferfollowed by 50 μL of diluted antibody solution were added to each well.Finally, 50 μL of test samples, standards or blanks were added per well.Plates were sealed and shaken at 450 rpm for 2 hrs. At the end of theincubation period the contents of the plate were removed, and the wellswere washed three times with 300 μL of diluted wash solution. 100 μL ofHRP solution was added to each well and incubated for 30 min underconstant shaking. The wells were emptied and washed four times with washsolution. 100 μL of the TMB substrate solution was added to each well.Plates were incubated in the dark for 30 min Finally, 100 μL of stopsolution was added to each well and within 5 min absorbance at 450 nmwas recorded. The values recorded for the blank controls were subtractedfrom the standards as well as sample values to eliminate background.Optical densities (OD) units were plotted using a 4-parameter fit forthe standard curve and were used to calculate the interferon titers inthe samples.

Human Tissue Specimens

Human skin specimens were collected as part of the Leiden LongevityStudy^(106,107) and were provided by the Leiden University MedicalCentre, Netherlands. Informed consent was obtained, and all protocolswere approved by the ethical committee of the Leiden University MedicalCentre. The samples were collected as 4 mm thickness full depth punchbiopsies, embedded in optimal cutting compound (OCT), flash frozen, andstored at −80° C. The investigators were blinded to everything exceptthe age and sex of the subjects. The OCT-embedded specimens werecryosectioned at 8 μm thickness using a Leica CM3050S cryomicrotome. Theslides were fixed with 4% PFA and 0.5% Triton X-100 in PBS (prewarmed to37° C.) for 20 min at room temperature. No further permeabilization wasperformed. Antibody incubation was preceded by a blocking step with 4%bovine serum albumin (BSA; fraction V, Thermo Fisher), 2% donkey serum,2% rabbit serum and 0.1% Triton X-100 in PBS for 1 hour at roomtemperature. Primary antibodies were diluted in the above blockingsolution (1:200) and incubated overnight at 4° C. with rocking in ahumidified chamber. The secondary antibodies (AlexaFluor 546 andAlexaFluor 647, Life Technologies) were also diluted in blockingsolution and incubated for 2 hrs at room temperature. Three 15 minwashing steps in PBS, containing 0.2% Triton X-100, followed eachantibody incubation. Nuclei were counterstained with 2 μg/mL DAPI inPBS, containing 0.2% Triton X-100, for 15 min. Stained slides weremounted with ProLong Antifade Mountant without DAPI (Life Technologies)and imaged on a Zeiss LSM 710 Confocal Laser Scanning Microscope. Az-series encompassing the full thickness of the tissue was collected foreach field. All microscope settings and exposure times were set tocollect images below saturation and were kept constant for all imagestaken in one experiment. Image analysis was performed using eitherCellProfiler software,¹⁰⁸ or ImageJ open source software from theNIH.¹⁰⁹ Nuclei were defined using the DAPI channel. Cell outlines weredefined by radially expanding the nuclear mask using the functionPropagate until an intensity threshold in the AlexaFluor 546 andAlexaFluor 647 channels was reached. The fluorescence intensity withinthese regions was then recorded in both channels. For each sample atotal of 200 nuclei were recorded in multiple fields. Mouse tissuesections were processed and analyzed in the same way as described above.

Mouse Issue Specimens

Total RNA was extracted from 50 mg of visceral adipose, small intestine,skeletal muscle, brown adipose or liver tissue by mincing followed byhomogenization in Trizol (Invitrogen) using a Power Gen 125 homogenizer(Fischer Scientific). After phase separation, the RNA in the aqueouslayer was purified using the Purelink RNA Mini kit (Invitrogen) withDNase I digestion. To assess gene expression by RT-qPCR 1 μg of totalRNA was reverse transcribed as described above. In each individualexperiment, all samples were processed in parallel and no blinding wasintroduced.

Imaging of whole-mount white adipose tissue followed the methoddescribed by Martinez-Santibañez et al. (2014).¹¹⁰ Briefly, whiteadipose tissue (visceral depot) were subdivided into 0.5-1 cm³ sizedpieces and incubated in 10 mL of fresh fixing buffer (1% PFA in PBS pH7.4) for 30 min at room temperature with gentle rocking. After threewashing steps with PBS, the tissue blocks were cut in six equal pieces.All subsequent incubations were performed in 2 mL cylindricalmicrocentrifuge tubes. Primary antibody incubation was preceded by ablocking step with 5% BSA, 0.1% Saponin in PBS for 30 min at roomtemperature. Primary antibodies were diluted in the above blockingsolution (1:200) and incubated overnight at 4° C. with gentle rocking.The secondary antibodies (AlexaFluor 546, AlexaFluor 594 and AlexaFluor647, Life Technologies) were also diluted in blocking solution andincubated for 2 hrs at room temperature. Three 10 min washing steps inPBS followed each antibody incubation. Antibody-independent staining ofnuclei and lipids was performed after immuno-staining: DAPI and BODIPY(Thermo Fisher) were diluted in PBS with 5% BSA and incubated withtissue specimens for 20 min followed by three washing steps as above.Stained samples were carefully placed on confocal-imaging optimized #1.5borosilicate glass chamber slides. A small drop of PBS prevented drying.Acquired images were analyzed as described above.

Co-staining of SA-β-Gal activity and ORF1 protein in liver sections wasperformed by staining for SA-β-Gal first as described.¹¹¹ Subsequently,samples underwent heat-induced epitope retrieval by steaming for 20 minin antigen retrieval buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween 20, pH9.0). Samples were then processed for immunofluorescence staining asabove (Human tissue specimens).

Kidney tissue preserved in OCT was cryosectioned, treated for 10 minwith 0.5% (w/v) periodic acid, then stained with periodic acid-Schiff's(PAS) reagent (Fisher Scientific, Cat. #SS32-500) for 10 min. Stainedtissue sections were mounted with Shandon Aqua Mount (Fisher Scientific,Cat. #14-390-5) and then imaged under bright field illumination.Glomerulosclerosis was scored as described.¹¹² Briefly, 40 glomeruli peranimal were assessed in a blinded fashion and assigned scores of 1-4:score of 1, <25% sclerosis; 2, 25-50% sclerosis; 3, 50-75% sclerosis;4, >75% sclerosis. The feature used to assess sclerosis was the strengthand pervasiveness of PAS-positive lesions within the glomeruli. Asexemplified in FIG. 4E, a sclerotic glomerulus is more shrunken andstains more intensely with PAS.

Quadriceps muscles were embedded in OCT, sectioned at 12 μm thicknessand mounted onto positively charged slides. Sections were stained withH&E (hematoxylin, 3 min followed by 30 eosin, sec). Mounted slides wereimaged on a Zeiss Axiovert 200M microscope equipped with a Zeiss MRC5color camera. To measure muscle fiber diameter, the shortest distanceacross ˜100 muscle fibers per animal was measured using ImageJ softwareas described.¹¹³ The Kolmogorov-Smirnov test was used to assess thestatistical significance of the difference between the resultingdistributions.

Statistical Treatments

Excel was used to perform general statistical analyses (means, s.d.,t-tests, etc.). R software for statistical computing (64-bit version3.3.2) was used for 1-way ANOVA and Tukey's multiple comparisonspost-hoc test. For consistency of comparisons significance in allfigures is denoted as follows: *P<0.05, **P<0.01. Sample sizes werebased on previously published experiments and previous experience inwhich differences were observed. No statistical test was used topre-determine sample size. No samples were excluded. All attempts atreplication were successful. There were no findings that were notreplicated or could not be reproduced. The nature and numbers of samplesanalyzed (defined as n) in each experiment are listed in the figurelegends. The number of independent experiments is also listed in figurelegends. The investigators were blinded when quantifyingimmunofluorescence results. Fields or sections of tissues forquantification were randomly selected and the scored, using methods asindicated for individual experiments. The investigators were alsoblinded when scoring glomerulosclerosis and muscle fiber diameter. ForRNA-seq and PCR array experiments the statistical treatments aredescribed under those sections (above).

Example 1 Activation of L1 and Interferon in Cellular Senescence

RTE activity can promote aberrant transcription, alternative splicing,insertional mutagenesis, DNA damage and genome instability.¹¹⁴RTE-derived sequences comprise up to two thirds of the human genome,¹¹⁵although the great majority were active millions of years ago and are nolonger intact. The only human RTE capable of autonomousretrotransposition is the long-interspersed element-1 (LINE-1, or L1).However, germline activity of L1 is a major source of human structuralpolymorphisms.¹¹⁶ Increasing evidence points to RTE activation in somecancers, in the adult brain, and during aging.^(117,118,119,120)Cellular defenses include heterochromatinization of the elements, smallRNA pathways that target the transcripts, and anti-viral innate immunitymechanisms.¹²¹ Somatic activation of RTEs with age is conserved in yeastand Drosophila and reducing RTE activity has beneficial effects.¹²²

As shown in FIG. 1A and FIGS. 6A-E, L1 transcription was activatedexponentially during replicative senescence (RS) of human fibroblasts,increasing 4-5-fold by 16 weeks after cessation of proliferation,referred to as late senescence. Multiple RT-qPCR primers were designedto detect evolutionarily recent L1 elements (L1HS-L1PA5; FIG. 1B, FIG.6H). Levels of L1 polyA+ RNA increased 4-5-fold in late senescent cells(RS) in the sense but not antisense direction throughout the entireelement (FIG. 1C). Long-range RT-PCR amplicons (FIG. 1B) were Sangersequenced to identify 224 elements dispersed throughout the genome; onethird (75, 33.5%) were L1HS, of which 19 (25.3%, 8.5% of total) wereintact (i.e., annotated to be free of ORF-inactivating mutations; FIGS.6F, 6G). 5′RACE were also performed with the same primers and found thatthe majority of L1 transcripts upregulated in senescent cells initiatedwithin or near the 5′UTR (FIG. 7 ).

L1 elements can stimulate an IFN-I response.¹²³ As shown in FIG. 1D andFIG. 6I, interferons IFN-α and IFN-β1 were induced to high levels inlate senescent cells. Cellular senescence proceeds through an early DNAdamage response phase followed by the SASP response.¹²⁴ Documented hereby the present data, is a third and even later phase, characterized bythe upregulation of L1 and an IFN-I response (FIG. 1E), which has notbeen previously noted, probably because most studies have focused onearlier times. Whole transcriptome RNA-seq analysis confirmed that theSASP and IFN-I responses are temporally distinct (FIG. 8 ). The latephase of L1 activation and IFN-I induction was also observed inoncogene-induced senescence (OIS) and stress-induced prematuresenescence (SIPS) (FIG. 1E).

Example 2 Mechanisms of L1 Activation

To explore how surveillance fails during senescence, three factors wereexamined: TREX1, RB1 and FOXA1. TREX1 is a 3′ exonuclease that degradesforeign invading DNAs and its loss has been associated with theaccumulation of cytoplasmic L1 cDNA.¹²⁵ As shown in FIG. 9A, theexpression of TREX1 was significantly decreased in senescent cells. RB1has been shown to bind to repetitive elements, including L1s, andpromote their heterochromatinization.¹²⁶ As shown in FIG. 2A, theexpression of RB1 declined strongly in senescent cells (RS) while thatof other RB family members (RBL1, RBL2) did not change (FIG. 9B). RB1enrichment in the 5′UTR of L1 elements was evident in proliferatingcells, decreased in early senescence and became undetectable at latertimes (FIG. 2A). This coincided with a decrease of H3K9me3 and H3K27me3marks in these regions (FIG. 9C).

To identify novel factors that interact with the L1 5′UTR, we examinedthe ENCODE ChIP-seq database and found that the pioneering transcriptionfactor FOXA1 binds to this region in several cell lines (FIG. 9D). FOXA1is upregulated in senescent cells.¹²⁷ As shown in FIG. 2B, FOXA1 boundto the central region of the L1 5′UTR. Using transcriptional reporters,we found that deletion of the FOXA1 binding site decreased both senseand antisense transcription from the L1 5′ UTR¹²⁸ (FIG. 9 e ). Hence,the observed misregulation of these three factors in senescent cellscould promote the activation of L1 by three additive mechanisms: loss ofRB1 by relieving heterochromatin repression, gain of FOXA1 by activatingthe L1 promoter, and loss of TREX1 by compromising the removal of L1cDNA.

Therefore, the effects of manipulating RB1, FOXA1 or TREX1 expression infully senescent cells were tested using lentiviral vectors (FIGS. 10A,10B). Ectopic expression of RB1 suppressed the elevated expression ofL1, IFN-α and IFN-β1 in senescent cells, while its knockdown furtherenhanced their expression (FIG. 2 ). RB1 overexpression also restoredits occupancy of the L1 5′UTR (FIG. 2C). Conversely, knockdown of FOXA1reduced its binding to the L1 5′UTR (FIG. 9F) and decreased theexpression of L1, IFN-α and IFN-β1, while overexpression of FOXA1increased L1, IFN-α and IFN-β1 levels (FIG. 2E). Congruent results werealso obtained by manipulating TREX1 (FIG. 2G). Hence, each of thesefactors had a tangible effect on regulating L1 and the IFN-I response insenescent cells.

Single or double interventions targeted at these factors elicited onlymodest changes in L1 and IFN-I expression in growing early passagecells. While some of these effects were statistically significant, theywere dwarfed by a triple intervention (3×) of RB1 and TREX1 knockdownscombined with FOXA1 overexpression, which resulted in a massiveinduction of L1 and IFN-I expression (FIGS. 2F, 9G-I, and 10C). Hence,in normal healthy cells, all three effectors have to be compromised toeffectively unleash L1.

Example 3 Consequences of L1 Activation

To assess IFN-I activation by L1 in more detail, we examined theexpression of 84 genes in this pathway using PCR arrays. We observed awidespread response, with the majority of the genes being upregulated(FIGS. 2H, 9J, 9K): 68% (57/84) were significantly upregulated insenescent cells, and 52% (44/84) were upregulated in 3× cells. Thesedata verify and further extend the RNA-seq transcriptomic analysis (FIG.8 ).

Some NRTIs developed against HIV have been found to also inhibit L1 RTactivity.¹²⁹ We also developed shRNAs against L1, two of which reducedtranscript levels by 40-50% and 70-90% in deeply senescent and 3× cells,respectively (FIG. 10G). ORF1 protein levels were correspondinglyreduced in deeply senescent cells (FIG. 5H). Finally, the shRNAs alsoreduced the retrotransposition of recombinant L1 reporter constructs(FIG. 10K).

Cells devoid of TREX1 display cytoplasmic L1 DNA, accumulation of whichcan be inhibited with NRTIs.¹³⁰ While lack of BrdU incorporation is acanonical feature of senescent cells (FIG. 68 ), longer term labelingrevealed DNA that was predominantly cytoplasmic and highly enriched forL1 sequences (FIGS. 11A, 11B). The synthesis of cytoplasmic L1 DNA couldbe almost completely blocked with the NRTI lamivudine (3TC) or shRNA toL1 (FIGS. 3A, 3C). An antibody to DNA:RNA hybrids detected a cytoplasmicsignal in senescent cells that largely colocalized with ORF1 protein andturned into a ssDNA signal following RNase digestion (FIG. 11C).Analysis of BrdU-labeled L1 sequences in senescent cells showed them tobe localized throughout the L1 element (FIGS. 11D, 11E). A relativeincrease of L1HS sequences in total cellular DNA can also be detected bya qPCR assay 6, 16.^(131,132) TC in the range of 7.5-10 μM completelyblocked this increase in senescent cells and also quenched the activityof a L1 retrotransposition reporter (FIGS. 10D, 10E).

L1 knockdown with shRNA or treatment of cells with 3TC significantlyreduced interferon levels, as well as reducing the IFN-I response morebroadly in both late senescent and 3× cells (FIGS. 3B, 12A). 3TC in therange of 7.5-10 μM optimally inhibited the IFN-I response, and was themost effective of 4 NRTIs tested (FIGS. 10F, 10J). The relativeefficacies of the NRTIs are consistent with their ability to inhibithuman L1 RT15. 3TC also antagonized the IFN-I response in other forms ofsenescence, OIS and SIPS (FIG. 3E).

Cells were passaged in the continuous presence of 3TC from theproliferative phase into deep senescence. 3TC did not significantlyaffect the timing of entry into senescence, induction of p21 or p16, orthe early SASP response, such as upregulation of IL-0 (FIGS. 3F, 12B).However, the magnitude of the later SASP response (such as induction ofCCL2, IL-6 and MMP3) was significantly dampened. Treatment with L1 shRNAalso reduced the expression levels of IL-6 and MMP3 in late senescentcells (FIG. 11F). Hence, while L1 activation and the ensuing IFN-Iresponse are relatively late in onset, they contribute importantly tothe mature SASP and proinflammatory phenotype of senescent cells.

3TC did not affect L1 transcript levels (FIG. 10I), suggesting that theINF-I response is triggered by L1 cDNA. As this model would predict,knockdown of the cytosolic DNA sensing pathway components, cGAS orSTING,¹³³ inhibited the IFN-I response in both late senescent and 3×cells (FIGS. 910L, 12C, 12D), and also downregulated the SASP responsein late senescent cells (FIG. 12E).

NRTIs alkyl-modified at the 5′ ribose position cannot be phosphorylatedand hence do not inhibit RT enzymes. However, they possess intrinsicanti-inflammatory activity by inhibiting P2X7-mediated events thatactivate the NLRP3 inflammasome pathway.¹³⁴ Tri-methoxy-3TC (K-9), at 10μM or 100 μM, did not inhibit the IFN-I response in either latesenescent or 3× cells (FIG. 12F). Hence, the effect of 3TC on the IFN-Ipathway requires RT inhibition. At high concentrations (100 μM), K-9 hadsome inhibitory activity on markers of inflammation (FIG. 12G).

To test the role of interferon signaling in SASP, the IFN-α/βs receptors(IFNAR1 and 2) were inactivated using CRISPR/Cas9. Effective ablation ofIFN-I signaling was achieved in both early passage and deep senescentcells (FIG. 10M). In both replicative and SIPS forms of senescence, lossof interferon signaling antagonized late (CCL2, IL-6, MMP3) but notearly (IL-1β) SASP markers (FIG. 3D). This further demonstrates thatIFN-I signaling contributes to the establishment of a full and matureSASP response in senescent cells.

Example 4 Activation of L1 in Human and Mouse Tissues

Activation of L1 expression in human cancers has been detected with anORF1 antibody.¹³⁵ The same reagent showed widespread ORF1 expression inboth senescent and 3× cells (FIGS. 8A, 8C, 8F). In skin biopsies ofnormal aged human individuals, we found that 10.7% of dermal fibroblastswere positive for the senescence marker p16, which is in the rangedocumented in aging primates¹³⁶ (FIGS. 13B, 13D, 13F, 13H). Some of thep16 positive dermal fibroblasts were also positive for ORF1 (10.3%).Notably, we never observed ORF1 in the absence of p16 expression. Wealso detected, at the single cell level, the presence of phosphorylatedSTAT1, consistent with the presence of interferon signaling in thetissue microenvironment¹³⁷ (FIGS. 138, 13E, 13G). Hence, a fraction ofsenescent cells in normal human individuals display activation of L1,consistent with these events accumulating during the progression ofsenescence.

We next examined mice and found that L1 mRNA was progressivelyupregulated with age in several tissues (FIG. 15G). The detected L1 RNAsequences were predominantly sense strand, represented throughout theelement, and all three active L1 families were detectable (FIGS. 11G,11H). At the protein level, the frequency of L1 Orf1 positive cellsincreased in tissues with age (FIG. 4A). Regions of Orf1 stainingcolocalized with senescence-associated β-galactosidase (SA-β-Gal)activity (FIG. 48 ). Several IFN-I response genes (Ifn-α, Irf7, Oas1),as well as pro-inflammatory and SASP markers (II-6, Mmp3, Pai1, alsoknown as Serpine1), were upregulated in tissues of old mice (FIGS. 4C,14 ). An increase in L1 expression and IFN-I response genes (Ifn-α,Oas1) were also observed in an experimentally-induced model of cellularsenescence (young animals subjected to sublethal irradiation; FIG. 4D).

Old animals (26 months) were treated for two-weeks with 3TC(administered in water at human therapeutic doses). We found a broad andsignificant downregulation the IFN-I response and alleviation of theSASP pro-inflammatory state (FIG. 4C; for the full dataset, see FIG. 14and Table 7). Expression of L1 mRNA and p16 was weakly downregulated,but in most cases, did not reach statistical significance. K-9 did notaffect either the IFN-I or SASP responses. Immunofluorescence analysisof tissue sections confirmed that senescent cells expressed SASP, andOrf1-expressing cells activated IFN-I signaling (FIGS. 15A-C). Treatmentwith 3TC significantly reduced both IFN-I and SASP, but not L1expression or the presence of senescent cells. Hence, NRTIs can becategorized as “senostatic” agents, to contrast them from “senolytic”treatments that remove senescent cells from tissues.^(138,139)

Decreased adipogenesis¹⁴⁰ and thermogenesis¹⁴¹ are features of naturalaging and both were increased in old animals by 2 weeks of 3TC treatment(FIGS. 15D-F). As shown in FIG. 4E, longer term treatments (from 20 to26 months of age) were effective at opposing several known phenotypes ofaging: (i) macrophage infiltration of tissues, a hallmark of chronicinflammation,^(142,143) (ii) glomerulosclerosis of the kidney,¹⁴⁴ and(iii) skeletal muscle atrophy.¹⁴⁵ Macrophage infiltration of whiteadipose was especially responsive, returning to youthful (5 month)levels with only 2 weeks of 3TC.

The activation of endogenous L1 elements and the ensuing robustactivation of an IFN-I response is a novel phenotype of senescent cells,including naturally-occurring senescent cells in tissues. This phenotypeevolves progressively during the senescence response and appears to bean important, but hitherto unappreciated component of SASP. We show thatthe expression of three regulators, RB1, FOXA1 and TREX1 changes duringsenescence, and that these changes are both sufficient and necessary toallow the transcriptional activation of L1s (FIG. 4G). Hence, multiplesurveillance mechanisms need to be defeated to unleash L1, whichunderscores the importance of keeping these elements repressed insomatic cells.

The activation of innate immune signaling, in response to L1 activationduring cellular senescence and aging, proceeds through theinterferon-stimulatory DNA (ISD) pathway. Cytoplasmic DNA can originatefrom several sources, such as mtDNA released from stressedmitochondria¹⁴⁶ or cytoplasmic chromatin fragments (CCF) released fromdamaged nuclei.^(147,148) The present results suggest that L1 cDNA is animportant inducer of IFN-I in senescent cells. Remarkably, NRTItreatment effectively antagonized not only the IFN-I response but, alsomore broadly, reduced age-associated chronic inflammation in multipletissues.

Sterile inflammation, also known as inflammaging, is a hallmark of agingand a contributing factor to many age-related diseases.^(149,150) Thepresent data indicates that activation of L1 elements (and possiblyother RTEs) promotes inflammaging, and that the L1 RT is a relevanttarget for the treatment of age-associated inflammation and disorders.

Example 5 Effects of Adefovir and Lamivudine on Senescence-InducedIncreases in L1 Sequence Abundance, Interferon Gene Expression, and SASPGene Expression

The effects of Adefovir and Lamivudine on senescence-induced increasesin L1 sequence abundance, interferon gene expression, and SASP geneexpression were assessed in human fibroblast cell lines.

L1 sequence abundance (copy number) were assessed in three differenthuman fibroblast cell lines: LF1, IMR90, WI38 using qPCR assays. Assayswere normalized to 5S rDNA abundance. The control (CTRL) wasnon-treated, early passage, proliferating cells. Drugs were appliedcontinuously, in medium, from a few passages before senescence, intosenescence and then into late senescence. The “senescent” samples wereharvested four months after onset of senescence. Both drugs weresupplemented at 5 μM in the medium. Red bar in “senescent” conditions:cultures without drugs at 4 months in senescence. As shown in FIG. 17A,L1 copy number increased in senescence in all three cell lines, and bothdrugs significantly prevented this increase, with Adefovir beingsomewhat more effective than Lamivudine.

The effects of 5 μM Adefovir and Lamivudine on interferon geneexpression were assessed in two cell lines (LF1 and IMR90) and twointerferon genes (IFN-α and IFN-#1) as performed in FIG. 17A, exceptthat the expression of the indicated genes was measured by RT-qPCR. Asshown in FIG. 17B, the high interferon gene expression in senescentcells was significantly reduced in all cases by both drugs, and againAdefovir was somewhat more effective than Lamivudine. The red bars arecultures without drugs at 4 months in senescence.

The effects of 5 μM Adefovir and Lamivudine on SASP gene expression wereassessed in one cell line (LF1) using two SASP genes (IL-6 and MMP3).CTRL was the expression in normal, pre-senescent, untreated cells. Asshown in FIG. 17C, an increase in expression with senescence (red bars)was observed, and in both cases, SASP gene expression was significantlydecreased with drug treatment, with Adefovir being somewhat moreeffective than Lamivudine.

Finally, the effects of high concentrations of Lamivudine andEmtricitabine (10 μM and 50 μM) on interferon gene expression (IFN-α andIFN-#1) were assessed in the LF1 cell line. These were done by passagingLF1 cells into senescence, keeping them in senescence for three months,adding the drugs, keeping the cells in the presence of the drugs for 1month, then harvesting at 4 months. Interferon gene expression wasassessed by RT-qPCR. The control (CTRL) was cells treated as above butwithout drugs. As shown in FIG. 17D, at 10 μM, Lamivudine decreasedIFN-I induction, and was somewhat more effective than Emtricitabine. At50 μM, Lamivudine actually induced an increase in the expression ofinterferons, even surpassing the levels seen in untreated cell. Thisincrease is believed to be caused by the toxicity of the drug at thesehigh levels. In contrast, Emtricitabine at 50 μM decreased theexpression of interferons, even below the decrease observed with 10 μM.

Accordingly, at high doses, the toxicity of RTIs can impair theirability to halt or block the harmful effects of senescent cells andtheir ability to prevent or reverse age-related inflammation anddisorders.

Example 6 Comparative Assessment of Several RTIs in a Dose-ResponseAssay of Inhibition of L1 Activity in Mouse and Human Cells

Eight RTI compounds were assessed for their ability to inhibit LINE-1(L1) activity in a mouse L1 retrotransposition assay: lamivudine (3TC);stavudine; emtricitabine; apricitabine; tenofovir disiproxil;censavudine; elvucitabine; and tenofovir. Three RTI compounds wereassessed in a human L1 retrotransposition assay: lamivudine (3TC);censavudine; and elvucitabine.

Mouse LINE-1 Retrotransposition Assay

The dual luciferase-encoding plasmid pYX016 containing a mouse L1element was described in Xie et al., 2011.¹⁵¹ Lamivudine (3TC),stavudine (d4T), emtricitabine, apricitabine, tenofovir disoproxil andtenofovir were purchased from AK Science. Elvucitabine was obtained bycustom synthesis. Censavudine was synthesized by Oncolys BioPharma. HeLacervical cancer cells were cultivated at 37° C. in a humidified 5% CO₂incubator in Dulbecco's Modified Eagle's Medium (DMEM)—high glucose,with 4500 mg/L glucose, L-glutamine, sodium pyruvate and sodiumbicarbonate (Sigma), supplemented with 10% of heat inactivated fetalbovine serum (Thermo Fisher).

Assays were performed as described in Xie et al., 2011¹⁵¹ with severalmodifications. The reporter assay was performed in 96 well white Opticalbottom plates. 6000 HeLa cells were seeded in each well 24 hours priortransfection and compound treatment. All compounds were resuspended inDMSO. Stock solution concentrations varied from 50 mM to 1.25 mMdepending of the solubility of the compound. Serial dilutions (1:3) wereprepared in DMSO. Ten different concentrations of each compound weretested in triplicate. Medium containing different concentrations of thecompounds were prepared by adding 2 μL of the compound dilution to 1 mLof the culture medium. The final concentration of DMSO in the medium was0.2%. FuGENE® HD transfection reagent (Promega) was used to transfectthe plasmid pYX016 into the cells. The transfection mix was prepared inOpiMEM (Thermo Fisher) using a reagent to DNA ratio of 3.5:1 accordingto manufacturer's instructions. Culture medium was removed from thecells and discarded. The transfection mix (5 μL) was mixed with thecompound containing medium (100 μL/well) and this was added onto thecells of each well. Cells were incubated for 48H at 37° C./5% CO₂.

Luciferase reporter activity was quantified with the Dual-Luciferase®Reporter Assay System (Promega) according to manufacturer's instructionsfor multiwell plates with the following modification: Cells were lyseddirectly on the multiwell plate with 30 μL of the passive lysis buffer(PLB) for 20 min at room temperature, with gentle shaking to ensurecomplete cell lysis (instead of 20 μL of PLB for 15 min). Firefly andRenilla luciferase signals were measured using a SpectraMax i3xMulti-Mode Microplate Reader. Integration times of 100 ms and 10 ms wereused to measure the Firefly and Renilla signals respectively. RelativeL1 activity was calculated as Firefly/Renilla*10,000. Dose responseinhibition data were fit to a four-parameter logistic equation usingnon-linear regression (using Graphpad Prism 8), to determine IC₅₀ valuesfor each inhibitor. The experiment was performed twice, independently.

Results

The inhibitory dose-response curves for the eight RTI compounds in thetwo independent experiments are provided in FIG. 19A and FIG. 18B,respectively. The IC₅₀ values are summarized in Table 10. Surprisingly,two RTI compounds, censavudine and elvucitabine, had much lower IC₅₀values (about 100 nM) than the other RTI compounds, thus displayingunexpected mouse L1 inhibitory activity.

Human LINE-1 Retrotransposition Assay

Given their unexpected ability to inhibit mouse L1 activity, censavudineand elvucitabine were tested for their ability to inhibit human L1activity. Lamivudine was also tested for comparison.

Assays for human L1 were performed in very similar manner to those formouse. pYX017 encoding a human LINE-1 sequence¹⁵¹ was used in place ofpYX016. Because the human construct yields a lower signal, compoundswere incubated with cells for 72 hours instead of 48 hours, and cellswere seeded at a density of 2000/well instead of 6000/well. In addition,the transfection reagent to DNA ratio was 2:1 instead of 3.5:1. Results

The inhibitory dose-response curves for the three RTI compounds areprovided in FIG. 19A-B and the IC₅₀ values are summarized in Table 11.Again, censavudine and elvucitabine displayed surprising human L1inhibitory activity (IC₅₀ about 100 nM) when compared to lamivudine(IC₅₀ over 800 nM).

Cell Viability Assay

As described above, the toxicity of RTIs could impair their ability tohalt or block the harmful effects of senescent cells and their abilityto prevent or reverse age-related inflammation and disorders. Thepotential toxicity of lamivudine (3TC); stavudine; emtricitabine;apricitabine; tenofovir disiproxil; censavudine; elvucitabine; andtenofovir was assessed in a cell viability assay at the doses used togenerate the L1 inhibitory dose-response curves.

HeLa cells were treated with the different concentrations of the eightRTI compounds for 48 hours. Cell viability was measured withCellTiter-Glo® luminescent cell viability assay (Promega®) and presentedas percentage of cell viability versus untreated cells. Staurosporine,known to induce cell death, was used as a control.

Results

As shown in FIG. 20 , the eight RTI compounds did not induce anysignificant levels of cell death in contrast to the control,staurosporine.

In summary, censavudine and elvucitabine both displayed an unexpectedability to inhibit mouse and human L1 activity (IC₅₀ about 100 nM)without inducing toxicity in the cell viability assay at doses up to 2μM.

In related experiments, islatravir and other compounds were tested forinhibition of retrotransposition activity of human LINE-1 in HeLa cellsaccording to the following procedure.

HeLa cervical cancer cells were cultivated at 37° C. in a humidified 5%CO₂ incubator in Dulbecco's Modified Eagle's Medium (DMEM)—high glucose,with 4500 mg/L glucose, L-glutamine, sodium pyruvate and sodiumbicarbonate (Sigma), supplemented with 10% of heat inactivated fetalbovine serum (Thermo Fisher).

Assays were performed using reporter plasmid pYX017 as described (Xie,et al., 2011) with several modifications. The reporter assay wasperformed in 96-well white optical bottom plates. HeLa cells were seededin wells 24 h prior to transfection and compound treatment so that cellswere approximately 30% confluent on the day of transfection. Differentcell plating densities were tested and a density of 2×10³ cells wasdetermined to be optimal.

Compounds were resuspended in DMSO. Serial dilutions (1:3) were preparedin DMSO. Medium containing different concentrations of the compoundswere prepared by adding 2 μl of the compound dilution to 1 ml of theculture medium. The final concentration of DMSO in the medium was 0.2%.

FuGENE® HD transfection reagent (Promega, E2311, Lot 382574 and Lot397842) was used to transfect the plasmids into the cells. Thetransfection reagent: DNA mixture was prepared in OpiMEM (Thermo Fisher)according to manufacturer's instructions. Different ratios oftransfection reagent to DNA were tested and a ratio of 3:1 wasdetermined to be optimal. Culture medium was removed from the cells anddiscarded. The transfection reagent DNA mixture (5 μl) was mixed withthe compound containing medium (100 μl/well) and this was added onto thecells of each well. Cells were incubated at 37° C./5% CO₂ for differentincubation time. A 72 h incubation time was determined to be optimal.

Luciferase reporter activity was quantified with the Dual-Luciferase®Reporter Assay System (Promega) according to manufacturer's instructionsfor multiwell plates except that cells were lysed directly on themultiwell plate with 30 μl of the passive lysis buffer (PLB) for 20 minat room temperature, with gentle shaking to ensure complete cell lysis.

Firefly and Renilla luciferase signals were measured using a SpectraMaxi3x Multi-Mode Microplate Reader. Integration times of 100 ms and 10 mswere used to measure the Firefly and Renilla signals respectively.Relative L1 activity is calculated as Firefly/Renilla*1000 orFirefly/Renilla*10,000. Dose response inhibition data were fit to a fourparameter logistic equation using non-linear regression (using GraphpadPrism 8), to determine IC₅₀ values for each inhibitor.

The results are provided in Table 12 and Table 13. Islatravir exhibitedunexpectedly better human LINE-1 inhibitor activity compared to theother reverse transcriptase inhibitory drugs tested. Also, islatravirpenetrated the blood brain barrier (BBB) and demonstrated intracellularlongevity following single oral doses in Rhesus macaque. See, Stoddardet al., Antimicrob. Agents Chemother. 59:4190-8 (2015). It was alsofound that intracellular half-life of the phosphorylated form ofislatravir in PBMCs was greater than 72 h. Example 7 IslatravirInhibitory Quotient (IQ)

The in vitro IC₅₀ for human LINE-1 in cell-based assay is 1.29×10⁻³ μM.See Table 12. The islatravir HIV WT IC₅₀=0.2×10⁻³ μM. Grobler et al,“MK-8591 POTENCY AND PK PROVIDE HIGH INHIBITORY QUOTIENTS AT LOW DOSESQD AND QW,” Conference on Retroviruses and Opportunistic Infections,Abstract Number 481, March 2019.

The steady state PBMC EFdA-TP Inhibitory Quotient at C_(avg) for HIV wasmodeled from single dose PK based on human PK parameters of EFdA(plasma) and EFdA-TP (intracellular). Levin, J., Conference Report,“Single Doses as Low as 0.5 mg of the Novel NRTTI MK-8591 Suppress HIVfor At Least Seven Days,” 9th IAS Conference on HIV Science; Paris,France; 23-26 Jul. 2017. The human plasma PK of EFdA (oral dose 0.5-30mg) shows dose-proportional oral clearance (31-45 L/hr) and proportionalincreases in volume of distribution that contribute to longer T-half atdoses >1 mg. Intracellular PK of EFdA-TP in human PBMC is doseproportional with a T-half of 78.5-128 h. The EFdA-TP IC₅₀ for HIV=49nM. The Plasma-Brain K_(app) (0.25) was determined in the non-humanprimate. Stoddart et al., Antimicrob Agents Chemother 59:4190-4198(2015).

These data were used to estimate the IQ for human LINE-1 in PBMC andbrain. Based upon steady-state the EFdA-TP intracellular concentrations,a daily dose of 2 mg of islatravir will have robust inhibitory activityfor human LINE-1 in both PBMCs (IQ=51) and brain (IQ=13). See Table 14.

Tables described in the present disclosure are provided below.

TABLE 1  List of primers used in PCR analysis₁ Number DesignationSequence Notes Primer MDL15UTRPRAF GCCAAGATGGCCGAATAGGALINE-1 5′UTR, positions 12-31, sense orientation². set 1 MDL15UTRPRARAAATCACCCGTCTTCTGCGTLINE-1 5′UTR, positions 64-83, antisense orientation². Amplicon A,Used in RT-qPCR experiments to assess expression of FIG. 1bL1Hs RNA. Also used in ChIP experiments. Primer MDL15UTRBFCGAGATCAAACTGCAAGGCGLINE-1 5′UTR, positions 402-421, sense orientation². set 2 MDL15UTRBRCCGGCCGCTTTGTTTACCTALINE-1 5′UTR, positions 454-473, antisense orientation². Amplicon B,Used in RT-qPCR experiments to assess expression of FIG. 1bL1Hs RNA. Also used in ChIP experiments. Primer MDL15UTRCFTAAACAAAGCGGCCGGGAA LINE-1 5′UTR, positions 474-492, sense orientation².set 3 MDL15UTRCR AGAGGTGGAGCCTACAGAGGLINE-1 5′UTR, positions 530-549, antisense orientation². Amplicon C,Used in RT-qPCR experiments to assess expression of FIG. 1bL1Hs RNA. Also used in ChIP experiments. Primer MDL15UTRDFAGAGAGCAGTGGTTCTCCCALINE-1 5′UTR, positions 619-638, sense orientation². set 4 MDL15UTRDRCAGTCTGCCCGTTCTCAGATLINE-1 5′UTR, positions 647-666, antisense orientation². Amplicon D,Used in RT-qPCR experiments to assess expression of FIG. 1bL1Hs RNA. Also used in ChIP experiments. Primer MDL10RF1FACCTGAAAGTGACGGGGAGALINE-1 ORF1, positions 1395-1414, sense orientation². set 5 MDL10RF1RCCTGCCTTGCTAGATTGGGGLINE-1 ORF1, positions 1508-1527, antisense orientation². Amplicon E,Used in RT-qPCR experiments to assess expression of FIG. 1bL1Hs RNA. Also used in ChIP experiments. Primer ORF2FCAAACACCGCATATTCTCACTCALINE-1 ORF2, positions 5731-5753, sense orientation². set 6 ORF2RCTTCCTGTGTCCATGTGATCTCALINE-1 ORF2, positions 5772-5794, antisense orientation². ORF2 probeAGGTGGGAATTGAAC-VIC Probe for TaqMan experiments. Amplicon F,ORF2F and ORF2R were used in SYBR Green RT-qPCR FIG. 1bexperiments to assess expression of L1Hs RNA, and inconjunction with the ORF2 probe in TaqMan qPCRexperiments on genomic DNA to determine relative copynumbers of L1Hs elements. Primers were developedand validated as described³. Primer 5SF CTCGTCTGATCTCGGAAGCTAAGRibosomal 5S RNA gene, sense orientation. set 7 5SR GCGGTCTCCCATCCAAGTACRibosomal 5S RNA gene, antisense orientation. 5S probeAGGGTCGGGCCTGG-6FAM Probe for TaqMan experiments.Used for internal normalization, in conjunction withprimer set 3, in TaqMan qPCR experiments on genomicDNA to determine relative copy numbers of L1Hs elements.Primers were developed and validated as described³. Primer GAPDHFWTTGAGGTCAATGAAGGGGTC GAPDH, sense orientation. set 8 GAPDHRVGAAGGTGAAGGTCGGAGTCA GAPDH, antisense orientation.Used for RT-qPCR of the mRNA of the GAPDH gene(NM_001256799). Primers span an intron. Used asthe internal normalizer in gene expression experiments. PrimerGAPDHINT4F CCAGGAGTGAGTGGAAGACAG Intron 4 of GAPDH, sense orientation.set 9 GAPDHINT4R CTAGTTGCCTCCCCAAAGCAIntron 4 of GAPDH, antisense orientation.Used as a negative control in ChIP experiments. Primer ACP1IFNAFWTTGATGGCAACCAGTTCCAG Interferon alpha consensus, sense orientation.set 10 ACP1IFNARV1 TCATCCCAAGCAGCAGATGAInterferon alpha consensus, antisense orientation. ACP1IFNARV2TGTTCCCAAGCAGCAGATGA Interferon alpha consensus, antisense orientation.Used in RT-qPCR experiments to assess the collectiveexpression of human interferon alpha genes. All 3primers were used in a single reaction. Primer MDIFNB1FWACGCCGCATTGACCATCTAT IFNB1, sense orientation. set 11 MDIFNB1RVGTCTCATTCCAGCCAGTGCT IFNB1, antisense orientation.Used for RT-qPCR of the mRNA of the IFNB1 gene (NM_002176). PrimerMDHSRB1AF ACTCTCACCTCCCATGTTGC RB1, sense orientation. set 12 MDHSRB1ARATCCGTGCACTCCTGTTCTG RB1, antisense orientation.Used for RT-qPCR of the mRNA of the RB1 gene(NM_000321). Primers span an intron. Primer MDHSP107AFCCAAGAAGCGCTCTGCTGTA RBL1, sense orientation. set 13 MDHSP107ARGCAGGGGATTCTGCATCACTA RBL1, antisense orientation.Used for RT-qPCR of the mRNA of the RBL1 gene(NM_0028955). Primers span an intron. Primer MDHSP130FAGTCGCCCACCCCTCAGAT RBL2, sense orientation. set 14 MDHSP130RTTCCCTCCAGCGTGTAGCTT RBL2, antisense orientation.Used for RT-qPCR of the mRNA of the RBL2 gene(NM_005611). Primers span an intron. Primer MDTREX1CDSFTCCCCTTCGGATCTTAACAC TREX1, sense orientation. set 15 MDTREX1CDSRCGAAAAAGATGAGGGTCTGC TREX1, antisense orientation.Used for RT-qPCR of the mRNA of the TREX1 gene(NM_007248). Primers span an intron. Primer MDHSFOXA1FGGAAGACCGGCCAGCTAGAG FOXA1, sense orientation. set 16 MDHSFOXA1RTGAAGGAGTAGTGGGGGTCC FOXA1, antisense orientation.Used for RT-qPCR of the mRNA of the FOXA1 gene(NM_D04496). Primers span an intron. Primer JJMMP1FW AGCCTTCCAACTCTGGAGMMP1, sense orientation. set 17 JJMMP1RV TAATGTMMP1, antisense orientation. CCGATGATCTCCCCTGACAAUsed for RT-qPCR of the mRNA of the MMP1 gene(NM_001145938). Primers span an intron. Primer JJMMP3FWCCCACCTTACATACAGGATTGTG MMP3, sense orientation. set 18 JJMMP3RV AMMP3, antisense orientation. CCCAGACTTTCAGAGCTTTCTCAUsed for RT-qPCR of the mRNA of the MMP3 gene(NM_002422). Primers span an intron. Primer JJIL6FWCACTGGCAGAAAACAACCTGAA IL6, sense orientation. set 19 JJIL6RVACCAGGCAAGTCTCCTCATTGA IL6, antisense orientation.Used for RT-qPCR of the mRNA of the IL6 gene(NM_000600). Primers span an intron. Primer JJIL8FWGTTTTTGAAGAGGGCTGAGAATT CXCL8 (IL8), sense orientation. set 20 JJIL8RV CCXCL8 (IL8), antisense orientation. CCCTACAACAGACCCACACAATAUsed for RT-qPCR of the mRNA of the CXCL8 gene C(NM_000584). Primers span an intron. Primer JJCCL2FWAAGACCATTGTGGCCAAGGA CCL2, sense orientation. set 21 JJCCL2RVTTCGGAGTTTGGGTTTGCT CCL2, antisense orientation.Used for RT-qPCR of the mRNA of the CCL2 gene(NM_002982). Primers span an intron. Primer JJCXCL2FWAGAATGGGCAGAAAGCTTGTCT CXCL2, sense orientation. set 22 JJCXCL2RVCCTTCTGGTCAGTTGGATTTGC CXCL2, antisense orientation.Used for RT-qPCR of the mRNA of the CXCL2 gene(NM_002089). Primers span an intron. Primer IL1BETAFWCGCCAGTGAAATGATGGCTTAT IL-1β, sense orientation. set 23 IL1BETARVCTGGAAGGAGCACTTCATCTGT IL-1β, antisense orientation.Used for RT-qPCR of the mRNA of the IL1B gene(NM_000576). Primers span an intron Primer MDIRF9FW CAACTGAGGCCCCCTTTCAAIRF9, sense orientation. set 24 MDIRF9RV CGCCCGTTGTAGATGAAGGTIRF9, antisense orientation.Used for RT-qPCR of the mRNA of the IRF9 gene(NM_006084). Primers span an intron Primer MDIRF7FW GGCTGGAAAACCAACTTCCGIRF7, sense orientation. set 25 MDIRF7RV GTTATCCCGCAGCATCACGAIRF7, antisense orientation.Used for RT-qPCR of the mRNA of the IRF7 gene(NM_001572). Primers span an intron Primer MDHSOAS1AFTGGAGACCCAAAGGGTTGGA OAS1, sense orientation. set 26 MDHSOAS1ARAGGAAGCAGGAGGTCTCACC OAS1, antisense orientation.Used for RT-qPCR of the mRNA of the OAS1 gene(NM_016816). Primers span an intron Primer HSCGASFWACGTGCTGTGAAAACAAAGAAG cGAS, sense orientation. set 27 HSCGASRVGTCCCACTGACTGTCTTGAGG cGAS, antisense orientation.Used for RT-qPCR of the mRNA of the MB21D1 gene(NM_138441). Primers span an intron Primer HSSTING1FWATATCTGCGGCTGATCCTGC STING, sense orientation. set 28 HSSTING1RVGGTCTGCTGGGGCAGTTTAT STING, antisense orientation.Used for RT-qPCR of the mRNA of the TMEM173 gene(NM_198282). Primers span an intron Primer TICDKN1AFWGAGACTCTCAGGGTCGAAAACG CDKN1A, sense orientation. set 29 TICDKN1ARVTTCCTGTGGGCGGATTAGG CDKN1A, antisense orientation.Used for RT-qPCR of the mRNA of the CDKN1A gene(NM_000389). Primers span an intron Primer TICDKN2AFWCGGAAGGTCCCTCAGACATC CDKN2A (p16), sense orientation. set 30 TICDKN2ARVCCCTGTAGGACCTTCGGTGA CDKN2A (p16), antisense orientation.Used for RT-qPCR of the mRNA of the CDKN2A gene(NM_000077). Primers span an intron Primer MGAPDHF CGGCCGCATCTTCTTGTGGapdh, sense orientation. set 31 MGAPDHR GTGACCAGGCGCCCAATAGapdh, antisense orientation.Used for RT-qPCR of the mRNA of the Gapdh gene(NM_008084.3). Primers span an intron. Used as theinternal normalizer in gene expression experiments. Primer MMHSP90AB1FCCACCACCCTGCTCTGTACTA Hsp90ab1, sense orientation. set 32 MMHSP90AB1RCCTCTCCATGGTGCACTTCC Hsp90ab1, antisense orientation.Used for RT-qPCR of the mRNA of the Hsp90ab1 gene(NM_008302). Primers span an intron. Used as secondarynormalizer in gene expression experiments. Primer MMGUSBFWCGTGACCTTTGTGAGCAACG Gusb, sense orientation. set 33 MMGUSBRVCTGCTCCATACTCGCTCTGG Gusb, antisense orientation.Used for RT-qPCR of the mRNA of the Gusb gene(NM_010368). Primers span an intron. Used as secondarynormalizer in gene expression experiments. Primer GBCONIFNAFTCTGATGCAGCAGGTGGG Interferon alpha consensus, sense orientation. set 34GBCONIFNAR AGGGCTCTCCAGACTTCTGCTCTInterferon alpha consensus, antisense orientation. GUsed in RT-qPCR experiments to assess the collectiveexpression of murine interferon alpha genes. Primerswere developed and validated as described⁴. Primer GBIRF7FWCTCCTGAGCGCAGCCTTG Irf7, sense orientation. set 35 GBIRF7RVGTTCTTACTGCTGGGGCCAT Irf7, antisense orientation.Used for RT-qPCR of the mRNA of the Irf7 gene(NM_016850). Primers span an intron. Primer MMOAS1BFTCTGCTTTATGGGGCTTCGG Oas1, sense orientation. set 36 MMOAS1BRTCGACTCCCATACTCCCAGG Oas1, antisense orientation.Used for RT-qPCR of the mRNA of the Irf9 gene(NM_001083925). Primers span an intron. Primer JKLINE1FWCTGCCTTGCAAGAAGAGAGC Murine LINE-1 5′UTR, positions 392-412, senseset 37 JKLINE1RV AGTGCTGCGTTCTGATGATG orientation. Amplicon W,Murine LINE-1 5′UTR, 596-616, antisense orientation. Ext. DataUsed in RT-qPCR experiments to assess expression of FIG. 6f.murine L1 RNA. Primer P16MouseF CCAGGGCCGTGTGCATCdkn2a, sense orientation. set 38 P16MouseR TACGTGAACGTTGCCCATCACdkn2a, antisense orientation.Used for RT-qPCR of the mRNA of the Cdkn2a gene(NM_009877). Primers span an intron. Primer GBMMP3FWGACTCAAGGGTGGATGCTGT Mmp3, sense orientation. set 39 GBMMP3RVCCAACTGCGAAGATCCACTG Mmp3, antisense orientation.Used for RT-qPCR of the mRNA of the Mmp3 gene(NM_0108209). Primers span an intron. Primer GBIL6FWCGGAGAGGAGACTTCACAGAGG II-6, sense orientation. set 40 GBIL6RV AII-6, antisense orientation. TTTCCACGATTTCCCAGAGAACAUsed for RT-qPCR of the mRNA of the II6 gene(NM_031168). Primers span an intron. Primer MMPAI1FWGGAAGGGCAACATGACCAGG Pai1, sense orientation. set 41 MMPAI1RVAGCTGCTCTTGGTCGGAAAG Pai1, antisense orientation.Used for RT-qPCR of the mRNA of the Pai1 gene(NM_008871). Primers span an intron. Primer APACACAFCTGGCTGCATCCATTATGTCA Acaca, sense orientation. set 42 APACACARTGGTAGACTGCCCGTGTGAA Acaca, antisense orientation.Used for RT-qPCR of the mRNA of the Acaca gene(NM_133360). Primers span an intron. Primer APFASNF CTGCGTGGCTATGATTATGGFasn, sense orientation. set 43 APFASNR AGGTTGCTGTCGTCTGTAGTFasn, antisense orientation.Used for RT-qPCR of the mRNA of the Fasn gene(NM_007988). Primers span an intron. Primer APSREBF1FACTTTTCCTTAACGTGGGCCT Srebf1, sense orientation. set 44 APSREBF1RCATCTCGGCCAGTGTCTGTT Srebf1, antisense orientation.Used for RT-qPCR of the mRNA of the Srebf1 gene(NM_Q11480). Primers span an intron. Primer APCEBPAFTTCGGGTCGCTGGATCTCTA C/EBPα, sense orientation. set 45 APCEBPARTCAAGGAGAAACCACCACGG C/EBPα, antisense orientation.Used for RT-qPCR of the mRNA of the C/EBPα gene (NM_007678). PrimerAPACACBF AGAAGCGAGCACTGCAAGGTTG Acacb, sense orientation. set 46APACACBR GGAAGATGGACTCCACCTGGTT Acacb, antisense orientation.Used for RT-qPCR of the mRNA of the Acacb gene(NM_133904). Primers span an intron. Primer APPPARGFTTCGCTGATGCACTGCCTAT Pparg, sense orientation. set 47 APPPARGRGGAATGCGAGTGGTCTTCCA Pparg, antisense orientation.Used for RT-qPCR of the mRNA of the Pparg gene(NM_011146). Primers span an intron. Primer MDMML1ORF1FATCTGTCTCCCAGGTCTGCT Murine LINE-1 ORF1, positions 1341-1360, senseset 48 MDMML1ORF1R TCCTCCGTTTACCTTTCGCC orientation. Amplicon X,Murine LINE-1 ORF1, positions 1460-1441, antisense Ext. Dataorientation. FIG. 6f.Used in RT-qPCR experiments to assess expression of murine L1 RNA.Primer MDMML1ORF2F GCTTCGGTGAAGTAGCTGGAMurine LINE-1 ORF2, positions 4786-4805, sense set 49 MDMML1ORF2RTTCGTTAGAGTCACGCCGAG orientation. Amplicon Y,Murine LINE-1 ORF2, positions 4939-4920, antisense Ext. Dataorientation. FIG. 6f.Used in RT-qPCR experiments to assess expression of murine L1 RNA.Primer MDMML13UTRF AGCCAAATGGATGGACCTGGMurine LINE-1 3′UTR, positions 6344-6363, sense set 50 MDMML13UTRRAAGGAGGGGCATAGTGTCCA orientation. Amplicon Z,Murine LINE-1 3′UTR, positions 6538-6519, antisense Ext. Dataorientation. FIG. 6f.Used in RT-qPCR experiments to assess expression of murine L1 RNA.Primer MDMML1TFF ATCCGGACCAGAGGACAGGMurine LINE-1 5′UTR sense orientation. set 51 MDMML1TFR ATGGCGACCGCTGCTGMurine LINE-1 5′UTR, antisense orientation.Primers amplify one of the polymorphisms of L1Tf(I-III) families. PrimerMDMML1MDNF AGAGAACCGGACCCAATCCA Murine LINE-1 5′UTR sense orientation.set 52 MDMML1MDNR GCTTGTGCCCCTACTCAGACMurine LINE-1 5′UTR, antisense orientation.Primers amplify one of the polymorphisms of L1MdN(I) families. PrimerMDMML1MDAF TCCCCACGGGATCCTAAGAC Murine LINE-1 5′UTR sense orientation.set 53 MDMML1MDAF CTCTGCAGGCAAGCTCTCTTMurine LINE-1 5′UTR, antisense orientation.Primers amplify one of the polymorphisms of L1MdA (IV-VII) families. ₁1All sequences are listed in the 5′->3′ orientation. Primer sets 1-30 arespecific for the listed human genes; primer sets 31-53 aremurine-specific. ²2 All LINE-1 positions are relative to the L1Hsconsensus sequence (Repbase, http://www.girinst.org/repbase/). ³3 SeeCoufal, N.G. et al. L1 retrotransposition in human neural progenitorcells. Nature 460, 1127-31 (2009). ⁴4 See Gautier, G. et al. A type Iinterferon autocrine-paracrine loop is involved in Toll-likereceptor-induced interleukin-12p70 secretion by dendritic cells. J. Exp.Med. 201,1435-46 (2005).

TABLE 2 List of antibodies Antibody Antibody species Vendor CatalogNumber Application GAPDH Rabbit Cell Signaling 5174 Loading control forimmunoblotting GAPDH Mouse Sigma G8795 Loading control forimmunoblotting p16 Mouse Santa Cruz sc-756 Immunoblotting p16 MouseSanta Cruz sc-56330 (JC8) Immunofluorescence p21 Rabbit Santa Cruzsc-397 Immunoblotting RB1 Mouse BD Biosciences 554136 Immunoblotting,ChIP TREX1 Rabbit Cell Signaling 12215 Immunoblotting FOXA1 Rabbit Abcamab170933 Immunoblotting FOXA1 Goat Abcam ab5089 ChIP Phospho-STAT1(Tyr701) Mouse Santa Cruz sc-8394 Immunofluorescence Phospho-STAT2(Tyr689) Mouse Millipore 07-224 Immunofluorescence STAT2 Rabbit CellSignaling 4594S Immunoblotting IRF7 Rabbit Abcam ab109255 ImmunoblottingIRF9 Rabbit Novus Bio NBP2-16991 Immunofluorescence BrdU Mouse BDBiosciences 555627 Immunofluorescence γ-H2AX Mouse Millipore 05-636Immunofluorescence F4/80 Rat Abcam ab6640 (CI:A3-1) ImmunofluorescencessDNA Mouse Enzo MAb F7-26 Immunofluorescence DNA-RNA hybrids MouseKerafast ENH001 (S9.6) Immunofluorescence LaminB1 Goat Santa CruzSc-6216 (C-20) Immunofluorescence IL-6 Rabbit Cell Signaling 12912Immunofluorescence Human LINE-1 ORF1 Rabbit Gift of K. H. Burns, JohnsHopkins¹ Immunofluorescence Mouse LINE-1 Orf1 Rabbit J. D. Boeke, abEA02(RabMAb clone Immunofluorescence NYU-2-1_2 ¹See Rodic, N. et al. Longinterspersed element-1 protein expression is a hallmark of many humancancers. Am. J. Pathol. 184, 1280-6 (2014).

TABLE 3 List of expressed L1 elements identified by long range RT-PCR IDChromosome Coordinates (element length) Family Count Intact L1-07 chr1463,115,693-63,121,722 (6,029 bp) L1HS 16 Yes L1-09 chrX11,934,270-11,940,496 (6,226 bp) L1HS 10 Yes L1-13 chr1766,596,080-66,602,096 (6,016 bp) L1HS 9 Yes L1-20 chr1470,546,288-70,552,339 (6,051 bp) L1HS 6 Yes L1-24 chr749,679,257-49,685,289 (6,032 bp) L1HS 5 Yes L1-25 chr421,158,389-21,164,420 (6,031 bp) L1HS 5 Yes L1-54 chr179,614,984-9,621,024 (6,040 bp) L1HS 3 Yes L1-55 chrX155,515,003-155,521,032 (6,029 bp) L1HS 2.125 Yes L1-58 chrX47,782,657-47,788,686 (6,029 bp) L1HS 2.125 Yes L1-59 chr1118,851,349-118,857,375 (6,026 bp) L1HS 2.125 Yes L1-62 chr169,583,477-9,589,517 (6,040 bp) L1HS 2.125 Yes L1-76 chr2166,987,450-166,993,486 (6,036 bp) L1HS 2 Yes L1-85 chr8128,451,975-128,458,006 (6,031 bp) L1HS 1.8 Yes L1-116 chr5104,517,585-104,523,614 (6,029 bp) L1HS 1 Yes L1-143 chr1180,865,798-180,871,853 (6,055 bp) L1HS 1 Yes L1-171 chr1098,781,926-98,787,958 (6,032 bp) L1HS 0.8 Yes L1-172 chr62,416,758-2,422,787 (6,029 bp) L1HS 0.8 Yes L1-180 chr3159,094,350-159,100,395 (6,045 bp) L1HS 1 Yes L1-218 chr1570,728,652-70,734,159 (5,507 bp) L1HS 1 Yes Sum: L1HS intact Counts:71.9 Individual elements: 19 L1-06 chr7 70,193,829-70,199,858 (6,029 bp)L1HS 18 L1-08 chr4 87,346,624-87,352,647 (6,023 bp) L1HS 11 L1-17 chr478,346,978-78,353,011 (6,033 bp) L1HS 7 L1-23 chr1813,975,361-13,980,770 (5,409 bp) L1HS 5 L1-31 chr9110,787,598-110,793,630 (6,032 bp) L1HS 4 L1-35 chr1196,218,871-196,224,903 (6,032 bp) L1HS 4 L1-37 chr3103,555,522-103,561,554 (6,032 bp) L1HS 4 L1-38 chr123,498,696-3,504,729 (6,033 bp) L1HS 4 L1-40 chr16 65,686,512-65,692,528(6,016 bp) L1HS 4 L1-41 chrX 50,018,977-50,025,006 (6,029 bp) L1HS 4L1-45 chr4 136,292,503-136,298,555 (6,052 bp) L1HS 4 L1-50 chr326,397,518-26,403,541 (6,023 bp) L1HS 3 L1-56 chr2023,422,609-23,428,641 (6,032 bp) L1HS 2.125 L1-57 chr1125,119,462-125,125,488 (6,026 bp) L1HS 2.125 L1-60 chr2011,629,280-11,635,312 (6,032 bp) L1HS 2.125 L1-61 chr1376,612,324-76,618,354 (6,030 bp) L1HS 2.125 L1-64 chr286,660,186-86,666,388 (6,202 bp) L1HS 2 L1-72 chr3132,945,507-132,951,535 (6,028 bp) L1HS 2 L1-80 chr5166,966,282-166,972,316 (6,034 bp) L1HS 2 L1-81 chr419,077,399-19,083,430 (6,031 bp) L1HS 2 L1-86 chr7 96,846,151-96,852,181(6,030 bp) L1HS 1.8 L1-87 chr2 230,336,570-230,342,014 (5,444 bp) L1HS1.8 L1-88 chr4 135,177,641-135,183,248 (5,607 bp) L1HS 1.8 L1-89 chr452,534,972-52,540,999 (6,027 bp) L1HS 1.8 L1-90 chr581,612,591-81,618,619 (6,028 bp) L1HS 2 L1-91 chr8128,890,989-128,897,136 (6,147 bp) L1HS 2 L1-96 chr1187,339,532-87,345,562 (6,030 bp) L1HS 2 L1-97 chr5177,771,244-177,777,273 (6,029 bp) L1HS 2 L1-98 chr5166,140,692-166,146,724 (6,032 bp) L1HS 2 L1-100 chr462,726,449-62,732,470 (6,021 bp) L1HS 1 L1-103 chr180,942,648-80,948,701 (6,053 bp) L1HS 1 L1-114 chr10105,775,021-105,781,052 (6,031 bp) L1HS 1 L1-115 chr534,144,346-34,150,370 (6,024 bp) L1HS 1 L1-133 chr7113,530,583-113,536,613 (6,030 bp) L1HS 1 L1-135 chr1180,330,072-180,336,104 (6,032 bp) L1HS 1 L1-136 chr1118,634,254-118,640,283 (6,029 bp) L1HS 1 L1-137 chrX54,117,700-54,123,731 (6,031 bp) L1HS 1 L1-138 chrX147,650,235-147,656,268 (6,033 bp) L1HS 1 L1-142 chr1187,217,153-187,223,183 (6,030 bp) L1HS 1 L1-144 chr1269,772,911-69,778,942 (6,031 bp) L1HS 1 L1-149 chr1239,622,999-239,629,024 (6,025 bp) L1HS 1 L1-150 chr1146,716,693-146,722,719 (6,026 bp) L1HS 1 L1-157 chr319,495,494-19,501,518 (6,024 bp) L1HS 1 L1-169 chr558,383,173-58,389,205 (6,032 bp) L1HS 0.8 L1-170 chr619,761,393-19,767,419 (6,026 bp) L1HS 0.8 L1-173 chr236,112,007-36,118,038 (6,031 bp) L1HS 0.8 L1-179 chr6112,700,246-112,706,279 (6,033 bp) L1HS 1 L1-181 chr3108,745,901-108,751,932 (6,031 bp) L1HS 1 L1-182 chr415,838,047-15,844,384 (6,337 bp) L1HS 1 L1-184 chr8135,872,363-135,878,417 (6,054 bp) L1HS 1 L1-187 chr1358,673,408-58,679,295 (5,887 bp) L1HS 1 L1-194 chr185,745,020-85,749,612 (4,592 bp) L1HS 1 L1-196 chr1582,882,394-82,888,420 (6,026 bp) L1HS 1 L1-197 chr1551,416,717-51,422,747 (6,030 bp) L1HS 1 L1-199 chr2 4,730,230-4,736,261(6,031 bp) L1HS 1 L1-212 chr3 54,393,823-54,400,093 (6,270 bp) L1HS 1Sum: L1HS non-intact Counts: 132.1 Individual elements: 56 L1-05 chr3141,764,155-141,770,160 (6,005 bp) L1PA2 24 L1-10 chr4164,097,249-164,103,279 (6,030 bp) L1PA2 9 L1-12 chr1761,106,730-61,112,789 (6,059 bp) L1PA2 9 L1-18 chr2211,955,882-11,962,029 (6,147 bp) L1PA2 6 L1-22 chr1267,896,709-67,902,725 (6,016 bp) L1PA2 5 L1-27 chr3175,781,211-175,786,413 (5,202 bp) L1PA2 5 L1-30 chr1373,637,028-73,643,060 (6,032 bp) L1PA2 5 L1-33 chr498,316,999-98,322,499 (5,500 bp) L1PA2 4 L1-34 chr3178,856,449-178,862,480 (6,031 bp) L1PA2 4 L1-39 chr1169,251,062-169,257,094 (6,032 bp) L1PA2 4 L1-42 chr3119,000,971-119,006,991 (6,020 bp) L1PA2 4 L1-53 chr5106,424,883-106,430,882 (5,999 bp) L1PA2 3 L1-66 chr1486,095,435-86,101,470 (6,035 bp) L1PA2 2 L1-73 chr465,992,800-65,998,803 (6,003 bp) L1PA2 2 L1-74 chr1099,149,238-99,155,259 (6,021 bp) L1PA2 2 L1-75 chr172,826,947-72,833,271 (6,324 bp) L1PA2 2 L1-77 chr732,461,029-32,467,034 (6,005 bp) L1PA2 2 L1-83 chr367,082,246-67,088,304 (6,058 bp) L1PA2 2 L1-101 chr12102,826,678-102,832,665 (5,987 bp) L1PA2 1 L1-105 chr2025,326,450-25,331,184 (4,734 bp) L1PA2 1 L1-107 chr1350,978,555-50,984,571 (6,016 bp) L1PA2 1 L1-108 chr4102,204,431-102,210,459 (6,028 bp) L1PA2 1 L1-111 chrX113,084,049-113,090,072 (6,023 bp) L1PA2 1 L1-118 chr219,333,222-9,339,374 (6,152 bp) L1PA2 1 L1-123 chr2129,037,643-129,043,675 (6,032 bp) L1PA2 1 L1-125 chr585,454,310-85,460,328 (6,018 bp) L1PA2 1 L1-128 chr5 4,611,929-4,617,955(6,026 bp) L1PA2 1 L1-130 chr16 48,768,072-48,774,104 (6,032 bp) L1PA2 1L1-131 chr6 162,989,238-162,995,263 (6,025 bp) L1PA2 1 L1-132 chr868,358,979-68,364,479 (5,500 bp) L1PA2 1 L1-134 chr565,160,518-65,166,549 (6,031 bp) L1PA2 1 L1-140 chr4119,273,614-119,279,671 (6,057 bp) L1PA2 1 L1-145 chr324,088,959-24,094,992 (6,033 bp) L1PA2 1 L1-151 chr12108,993,448-108,999,474 (6,026 bp) L1PA2 1 L1-164 chr1741,200,368-41,206,401 (6,033 bp) L1PA2 1 L1-167 chr3118,910,744-118,916,772 (6,028 bp) L1PA2 1 L1-175 chr2197,687,033-197,693,062 (6,029 bp) L1PA2 1 L1-177 chrX154,935,536-154,941,561 (6,025 bp) L1PA2 1 L1-186 chr683,797,243-83,803,271 (6,028 bp) L1PA2 1 L1-188 chr887,621,331-87,626,401 (5,070 bp) L1PA2 1 L1-189 chr11100,649,611-100,655,672 (6,061 bp) L1PA2 1 L1-198 chr1554,904,988-54,911,051 (6,063 bp) L1PA2 1 L1-202 chr2153,781,069-153,787,086 (6,017 bp) L1PA2 1 L1-209 chr185,923,568-85,927,951 (4,383 bp) L1PA2 1 L1-211 chr2156,251,019-156,257,086 (6,067 bp) L1PA2 1 L1-215 chr4132,940,946-132,946,970 (6,024 bp) L1PA2 1 L1-221 chrX50,056,644-50,062,676 (6,032 bp) L1PA2 1 L1-223 chrX113,337,323-113,343,354 (6,031 bp) L1PA2 1 Sum: L1PA2 Counts: 124Individual elements: 48 L1-03 chr19 42,768,603-42,774,543 (5,940 bp)L1PA3 31 L1-04 chr8 55,046,402-55,052,502 (6,100 bp) L1PA3 28 L1-11chr12 118,657,390-118,663,531 (6,141 bp) L1PA3 9 L1-15 chr1176,307,883-176,313,876 (5,993 bp) L1PA3 7 L1-19 chr4175,016,241-175,022,252 (6,011 bp) L1PA3 6 L1-26 chr1849,212,269-49,218,417 (6,148 bp) L1PA3 5 L1-29 chr1122,646,426-22,652,446 (6,020 bp) L1PA3 5 L1-36 chr222,980,654-22,984,410 (3,756 bp) L1PA3 4 L1-44 chr10109,602,039-109,607,567 (5,528 bp) L1PA3 4 L1-46 chr763,728,965-63,735,094 (6,129 bp) L1PA3 4 L1-48 chr1438,631,448-38,637,485 (6,037 bp) L1PA3 4 L1-49 chr1942,743,461-42,749,399 (5,938 bp) L1PA3 4 L1-52 chr4123,632,778-123,638,935 (6,157 bp) L1PA3 3 L1-65 chr296,375,482-96,381,632 (6,150 bp) L1PA3 2 L1-69 chr1100,662,482-100,668,126 (5,644 bp) L1PA3 2 L1-70 chr525,077,154-25,083,186 (6,032 bp) L1PA3 2 L1-78 chr1278,502,931-78,509,088 (6,157 bp) L1PA3 2 L1-82 chr680,844,240-80,850,277 (6,037 bp) L1PA3 2 L1-84 chr869,015,505-69,021,651 (6,146 bp) L1PA3 2 L1-94 chr488,916,060-88,922,204 (6,144 bp) L1PA3 2 L1-104 chr4142,249,845-142,255,868 (6,023 bp) L1PA3 1 L1-110 chr324,276,690-24,282,186 (5,496 bp) L1PA3 1 L1-113 chr4119,061,402-119,067,563 (6,161 bp) L1PA3 1 L1-117 chr1210,343,959-10,349,500 (5,541 bp) L1PA3 1 L1-121 chr3112,658,647-112,664,665 (6,018 bp) L1PA3 1 L1-124 chr968,274,827-68,280,864 (6,037 bp) L1PA3 1 L1-126 chr575,792,130-75,798,187 (6,057 bp) L1PA3 1 L1-139 chr1131,540,511-31,546,645 (6,134 bp) L1PA3 1 L1-141 chr3145,924,279-145,930,431 (6,152 bp) L1PA3 1 L1-147 chr891,887,863-91,893,862 (5,999 bp) L1PA3 1 L1-148 chrX94,858,876-94,864,901 (6,025 bp) L1PA3 1 L1-152 chr1855,886,913-55,892,940 (6,027 bp) L1PA3 1 L1-154 chr9125,006,549-125,012,715 (6,166 bp) L1PA3 1 L1-156 chr671,826,216-71,832,235 (6,019 bp) L1PA3 1 L1-158 chr891,191,345-91,197,369 (6,024 bp) L1PA3 1 L1-159 chr1255,586,438-55,592,480 (6,042 bp) L1PA3 1 L1-165 chr297,521,921-97,528,067 (6,146 bp) L1PA3 1 L1-166 chr6126,670,052-126,676,070 (6,018 bp) L1PA3 1 L1-174 chr1280,143,556-80,149,577 (6,021 bp) L1PA3 1 L1-176 chr1251,562,132-51,568,340 (6,208 bp) L1PA3 1 L1-178 chr917,536,062-17,542,081 (6,019 bp) L1PA3 1 L1-183 chr1280,074,170-80,080,329 (6,159 bp) L1PA3 1 L1-185 chr648,733,849-48,740,059 (6,210 bp) L1PA3 1 L1-190 chr482,171,911-82,177,138 (5,227 bp) L1PA3 1 L1-191 chr1532,045,176-32,051,279 (6,103 bp) L1PA3 1 L1-192 chr1771,351,460-71,357,401 (5,941 bp) L1PA3 1 L1-193 chr599,339,116-99,345,270 (6,154 bp) L1PA3 1 L1-195 chr5122,509,114-122,515,140 (6,026 bp) L1PA3 1 L1-200 chr1035,855,169-35,861,196 (6,027 bp) L1PA3 1 L1-201 chr1231,159,575-31,165,595 (6,020 bp) L1PA3 1 L1-203 chr1437,766,297-37,772,332 (6,035 bp) L1PA3 1 L1-206 chr1456,013,511-56,019,543 (6,032 bp) L1PA3 1 L1-210 chr295,866,030-95,872,191 (6,161 bp) L1PA3 1 L1-214 chr494,835,259-94,841,286 (6,027 bp) L1PA3 1 L1-217 chr784,049,212-84,055,231 (6,019 bp) L1PA3 1 L1-219 chr1612,069,684-12,075,804 (6,120 bp) L1PA3 1 L1-222 chrX55,680,654-55,686,684 (6,030 bp) L1PA3 1 L1-224 chrX149,432,338-149,438,331 (5,993 bp) L1PA3 1 Sum: L1PA3 Counts: 166Individual elements: 58 L1-01 chr18 9,165,216-9,171,138 (5,922 bp) L1PA446 L1-14 chr3 29,978,961-29,984,962 (6,001 bp) L1PA4 7 L1-16 chr683,330,453-83,336,586 (6,133 bp) L1PA4 7 L1-21 chr6139,217,130-139,223,263 (6,133 bp) L1PA4 6 L1-28 chr849,874,774-49,880,758 (5,984 bp) L1PA4 5 L1-32 chr5176,077,678-176,083,819 (6,141 bp) L1PA4 4 L1-43 chr533,095,404-33,101,548 (6,144 bp) L1PA4 4 L1-47 chr448,864,081-48,870,233 (6,152 bp) L1PA4 4 L1-51 chr5130,779,991-130,786,158 (6,167 bp) L1PA4 3 L1-63 chr67,942,696-7,948,800 (6,104 bp) L1PA4 2 L1-71 chr7 56,505,920-56,512,049(6,129 bp) L1PA4 2 L1-79 chr10 93,517,474-93,523,652 (6,178 bp) L1PA4 2L1-92 chr21 9,182,589-9,188,720 (6,131 bp) L1PA4 2 L1-93 chr4173,772,935-173,779,072 (6,137 bp) L1PA4 2 L1-95 chr1125,011,752-125,017,885 (6,133 bp) L1PA4 2 L1-99 chr990,682,773-90,689,210 (6,437 bp) L1PA4 2 L1-102 chr1064,014,843-64,021,048 (6,205 bp) L1PA4 1 L1-112 chr1576,776,415-76,782,548 (6,133 bp) L1PA4 1 L1-119 chr1211,373,698-11,379,329 (5,631 bp) L1PA4 1 L1-120 chr1924,012,879-24,018,978 (6,099 bp) L1PA4 1 L1-122 chr399,031,289-99,037,430 (6,141 bp) L1PA4 1 L1-129 chr1015,664,019-15,669,631 (5,612 bp) L1PA4 1 L1-146 chr490,478,877-90,483,706 (4,829 bp) L1PA4 1 L1-153 chrX76,878,709-76,885,597 (6,888 bp) L1PA4 1 L1-155 chr1843,282,426-43,288,579 (6,153 bp) L1PA4 1 L1-160 chr1920,486,826-20,493,102 (6,276 bp) L1PA4 1 L1-161 chr1200,181,868-200,188,010 (6,142 bp) L1PA4 1 L1-162 chr11100,034,468-100,040,624 (6,156 bp) L1PA4 1 L1-163 chr2029,365,560-29,370,431 (4,871 bp) L1PA4 1 L1-168 chr578,706,259-78,712,394 (6,135 bp) L1PA4 1 L1-204 chr183,178,675-83,184,836 (6,161 bp) L1PA4 1 L1-205 chr339,366,145-39,372,264 (6,119 bp) L1PA4 1 L1-208 chr1845,793,571-45,799,710 (6,139 bp) L1PA4 1 L1-216 chr539,030,830-39,036,977 (6,147 bp) L1PA4 1 Sum: L1PA4 Counts: 118Individual elements: 34 L1-02 chr5 80,840,410-80,845,999 (5,589 bp)L1PA5 36 L1-67 chr12 370,113-376,219 (6,106 bp) L1PA5 2 L1-68 chr1195,811,406-95,816,742 (5,336 bp) L1PA5 2 L1-106 chr10109,822,318-109,828,350 (6,032 bp) L1PA5 1 L1-109 chrX12,102,927-12,109,072 (6,145 bp) L1PA5 1 L1-127 chr459,474,627-59,479,488 (4,861 bp) L1PA5 1 L1-207 chr1599,827,462-99,833,366 (5,904 bp) L1PA5 1 L1-213 chr3146,434,278-146,440,207 (5,929 bp) L1PA5 1 L1-220 chr1835,720,595-35,726,712 (6,117 bp) L1PA5 1 Sum: L1PA5 Counts: 46Individual elements: 9 Individual Summary Counts % elements % L1HSintact 71.9 10.9 19 8.5 L1HS non-intact 132.1 20.1 56 25.0 L1PA2 12418.8 48 21.4 L1PA3 166 25.2 58 25.9 L1 PA4 118 17.9 34 15.2 L1PA5 46 7.09 4.0 Sum: 658 100.0 224 100.0

TABLE 4 Gene list used in GSEA for IFN-I (50 genes) Gene Symbol GeneName ADAR adenosine deaminase, RNA specific BST2 bone marrow stromalcell antigen 2 CASP1 caspase 1 CAV1 caveolin 1 CXCL10 C-X-C motifchemokine ligand 10 DDX58 DExD/H-box helicase 58 EIF2AK2 eukaryotictranslation initiation factor 2 alpha kinase 2 IFI16 interferon gammainducible protein 16 IFI27 interferon alpha inducible protein 27 IFI30IFI30, lysosomal thiol reductase IFI6 interferon alpha inducible protein6 IFIH1 interferon induced with helicase C domain 1 IFIT1 interferoninduced protein with tetratricopeptide repeats 1 IFIT2 interferoninduced protein with tetratricopeptide repeats 2 IFIT3 interferoninduced protein with tetratricopeptide repeats 3 IFITM1 interferoninduced transmembrane protein 1 IFITM2 interferon induced transmembraneprotein 2 IFITM3 interferon induced transmembrane protein 3 IFNA1interferon alpha 1 IFNA2 interferon alpha 2 IFNA4 interferon alpha 4IFNAR1 interferon alpha and beta receptor subunit 1 IFNAR2 interferonalpha and beta receptor subunit 2 IFNB1 interferon beta 1 IFNEinterferon epsilon IFNW1 interferon omega 1 IRF1 interferon regulatoryfactor 1 IRF2 interferon regulatory factor 2 IRF3 interferon regulatoryfactor 3 IRF5 interferon regulatory factor 5 IRF7 interferon regulatoryfactor 7 IRF9 interferon regulatory factor 9 ISG15 ISG15 ubiquitin-likemodifier ISG20 interferon stimulated exonuclease gene 20 JAK1 Januskinase 1 JAK2 Janus kinase 2 MAL mal, T-cell differentiation protein METMET proto-oncogene, receptor tyrosine kinase MNDA myeloid cell nucleardifferentiation antigen MX1 MX dynamin like GTPase 1 MX2 MX dynamin likeGTPase2 MYD88 myeloid differentiation primary response 88 NOS2 nitricoxide synthase 2 OAS1 2′-5′-oligoadenylate synthetase 1 OAS22′-5′-oligoadenylate synthetase 2 STAT1 signal transducer and activatorof transcription 1 STAT2 signal transducer and activator oftranscription 2 STAT3 signal transducer and activator of transcription 3TMEM173 transmembrane protein 173 TNFSF10 tumor necrosis factorsuperfamily member 10

TABLE 5 GSEA analysis of KEGG pathways comparing early passage with early senescence UPREGULATED FROM EARLY PASSAGE (EP) TO EARLY SENESCENCE (SEN-E)PATHWAY NAME SIZE ES NES NOM p-val FDR p-val KEGG_ASTHMA 28 0.72371.7864 <1.00E−03 <1.00E−02 KEGG_GRAFT_VERSUS_HOST_DISEASE 35 0.68521.7654 <1.00E−03 <1.00E−02 KEGG_CELL_ADHESION_MOLECULES_CAMS 127 0.55781.7538 <1.00E−03 <1.00E−02 KEGG_OLFACTORY_TRANSDUCTION 367 0.4965 1.7468<1.00E−03 <1.00E−02 KEGG_TYPE_I_DIABETES_MELLITUS 41 0.6457 1.7140<1.00E−03 <1.00E−02 KEGG_LYSOSOME 119 0.5388 1.6878 <1.00E−03 <1.00E−02KEGG_COMPLEMENT_AND_COAGULATION_CASCADES 67 0.5786 1.6854 <1.00E−03<1.00E−02 KEGG_ECM_RECEPTOR_INTERACTION 84 0.5123 1.5487 <1.00E−03<1.00E−02 KEGG_LEISHMANIA_INFECTION 68 0.5688 1.6559  1.79E−03  1.51E−02CUSTOM_SASP 85 0.5471 1.6517  1.80E−03  1.51E−02 KEGG_PRION_DISEASES 340.6326 1.6397  1.84E−03  1.51E−02KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION 254 0.4561 1.5705  1.62E−03 1.51E−02 KEGG_TOLL_LIKE_RECEPTOR_SIGNALING_PATHWAY 99 0.4868 1.4920 1.73E−03  1.51E−02 KEGG_AUTOIMMUNE_THYROID_DISEASE 48 0.5605 1.5336 3.77E−03  2.66E−02 KEGG_ENDOCYTOSIS 174 0.4365 1.4427  5.21E−03 3.49E−02 KEGG_VIRAL_MYOCARDITIS 68 0.5313 1.5277  8.68E−03  5.47E−02KEGG_RIG_I_LIKE_RECEPTOR_SIGNALING_PATHWAY 67 0.5169 1.4977  9.06E−03 5.47E−02 KEGG_INTESTINALIMMUNE_NETWORK_FOR_ 45 0.5848 1.5670  1.08E−02 6.11E−02 IGA_PRODUCTION KEGG_HYPERTROPHIC_CARDIOMYOPATHY_HCM 83 0.48141.4387  1.08E−02  6.11E−02 KEGG_NEUROACTIVE_LIGAND_RECEPTOR_INTERACTION270 0.3779 1.2928  1.12E−02  6.14E−02KEGG_ANTIGEN_PROCESSING_AND_PRESENTATION 73 0.5096 1.4914  1.28E−02 6.81E−02 KEGG_EPITHELIAL_CELL_SIGNALING_IN_ 67 0.5106 1.5038  1.64E−02 8.00E−02 HELICOBACTER_PYLORI_INFECTIONKEGG_STARCH_AND_SUCROSE_METABOLISM 45 0.5382 1.4727  1.68E−02  8.00E−02KEGG_MAPK_SIGNALING_PATHWAY 263 0.3751 1.2821  1.64E−02  8.00E−02KEGG_ASCORBATE_AND_ALDARATE_METABOLISM 21 0.6766 1.5631  2.06E−02 9.32E−02 KEGG_NOD_LIKE_RECEPTOR_SIGNALING_PATHWAY 60 0.5046 1.4370 2.16E−02  9.54E−02 KEGG_ABC_TRANSPORTERS 44 0.5440 1.4685  2.51E−02 1.08E−01 KEGG_CYTOSOLIC_DNA_SENSING_PATHWAY 52 0.5357 1.4752  2.64E−02 1.11E−01 KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY 123 0.42181.3254  2.83E−02  1.16E−01 KEGG_OTHER_GLYCAN_DEGRADATION 16 0.69671.5277  3.13E−02  1.24E−01 KEGG_ALANINE_ASPARTATE_AND_GLUTAMATE_ 320.5745 1.4414  3.22E−02  1.24E−01 METABOLISMKEGG_HEMATOPOIETIC_CELL_LINEAGE 85 0.4547 1.3563  3.42E−02  1.27E−01KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_ 22 0.6172 1.4675  3.59E−02 1.27E−01 CHONDROITIN_SULFATE KEGG_ARRHYTHMOGENIC_RIGHT_VENTRICULAR_ 740.4733 1.3664  3.54E−02  1.27E−01 CARDIOMYOPATHY_ARVCKEGG_DILATED_CARDIOMYOPATHY 90 0.4453 1.3480  3.55E−02  1.27E−01KEGG_ALLOGRAFT_REJECTION 35 0.5607 1.4452  3.92E−02  1.36E−01KEGG_CALCIUM_SIGNALING_PATHWAY 173 0.3956 1.2886  4.46E−02  1.52E−01KEGG_REGULATION_OF_ACTIN_CYTOSKELETON 210 0.3708 1.2426  4.93E−02 1.62E−01 KEGG_STEROID_HORMONE_BIOSYNTHESIS 49 0.4982 1.3661  5.14E−02 1.66E−01 KEGG_LINOLEIC_ACID_METABOLISM 29 0.5664 1.3972  5.98E−02 1.90E−01 KEGG_FOCAL_ADHESION 197 0.3646 1.2184  6.69E−02  2.09E−01KEGG_LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION 115 0.4016 1.2474  7.64E−02 2.34E−01 KEGG_RETINOL_METABOLISM 56 0.4497 1.2695  9.09E−02  2.70E−01KEGG_CHEMOKINE_SIGNALING_PATHWAY 183 0.3602 1.1945  9.02E−02  2.70E−01KEGG_VASCULAR_SMOOTH_MUSCLE_CONTRACTION 112 0.3902 1.2155  9.52E−02 2.78E−01 KEGG_PATHOGENIC_ESCHERICHIA_COLI_INFECTION 53 0.4475 1.2529 1.16E−01  3.33E−01 KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_ 15 0.59411.2772  1.73E−01  4.70E−01 GANGLIO_SERIES KEGG_SPHINGOLIPID_METABOLISM36 0.4710 1.2155  1.79E−01  4.70E−01 KEGG_REGULATION_OF_AUTOPHAGY 320.4748 1.2109  1.75E−01  4.70E−01 KEGG_ARACHIDONIC_ACID_METABOLISM 580.4212 1.1842  1.78E−01  4.70E−01 KEGG_MELANOMA 71 0.4044 1.1820 1.76E−01  4.70E−01 KEGG_METABOLISM_OF_XENOBIOTICS_BY_ 62 0.4067 1.1816 1.88E−01  4.86E−01 CYTOCHROME_P450 KEGG_AXON_GUIDANCE 127 0.3540 1.1188 2.01E−01  5.12E−01 KEGG_SNARE_INTERACTIONS_IN_VESICULAR_TRANSPORT 380.4560 1.1899  2.08E−01  5.23E−01KEGG_NICOTINATE_AND_NICOTINAMIDE_METABOLISM 22 0.5028 1.1791  2.20E−01 5.31E−01 KEGG_TASTE_TRANSDUCTION 50 0.4249 1.1650  2.19E−01  5.31E−01KEGG_NITROGEN_METABOLISM 23 0.5036 1.1769  2.24E−01  5.33E−01KEGG_PPAR_SIGNALING_PATHWAY 69 0.3797 1.1045  2.63E−01  5.77E−01KEGG_JAK_STAT_SIGNALING_PATHWAY 148 0.3340 1.0743  2.65E−01  5.77E−01KEGG_APOPTOSIS 86 0.3669 1.0996  2.67E−01  5.77E−01KEGG_DRUG_METABOLISM_OTHER_ENZYMES 44 0.4154 1.1191  2.81E−01  5.98E−01KEGG_DRUG_METABOLISM_CYTOCHROME_P450 63 0.3754 1.0737  2.93E−01 6.17E−01 KEGG_INOSITOL_PHOSPHATE_METABOLISM 54 0.3887 1.0905  3.04E−01 6.32E−01 KEGG_PHOSPHATIDYLINOSITOL_SIGNALING_SYSTEM 73 0.3662 1.0598 3.33E−01  6.77E−01 KEGG_GLYCOSPHINGOLIPID_BIOSYNTHESIS_LACTO_ 25 0.44341.0764  3.41E−01  6.86E−01 AND_NEOLACTO_SERIES KEGG_ALZHEIMERS_DISEASE153 0.3226 1.0359  3.57E−01  7.10E−01 CUSTOM_IFN-I 50 0.3781 1.0455 3.69E−01  7.18E−01 KEGG_FRUCTOSE_AND_MANNOSE_METABOLISM 34 0.39711.0438  3.81E−01  7.24E−01 KEGG_GLYCOSAMINOGLYCAN_BIOSYNTHESIS_ 260.4256 1.0321  3.84E−01  7.24E−01 HEPARAN_SULFATEKEGG_PANTOTHENATE_AND_COA_BIOSYNTHESIS 16 0.4687 1.0325  4.21E−01 7.67E−01 KEGG_N_GLYCAN_BIOSYNTHESIS 46 0.3798 1.0231  4.32E−01 7.67E−01 KEGG_GALACTOSE_METABOLISM 26 0.4132 1.0123  4.50E−01  7.83E−01KEGG_GLYCEROLIPID_METABOLISM 43 0.3679 0.9942  4.71E−01  8.12E−01KEGG_LONG_TERM_POTENTIATION 66 0.3408 0.9796  4.99E−01  8.36E−01KEGG_RENIN_ANGIOTENSIN_SYSTEM 17 0.4406 0.9772  4.95E−01  8.36E−01KEGG_MATURITY_ONSET_DIABETES_OF_THE_YOUNG 25 0.4094 0.9773  5.17E−01 8.41E−01 KEGG_WNT_SIGNALING_PATHWAY 149 0.3005 0.9739  5.21E−01 8.41E−01 KEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSIS 92 0.3151 0.9621 5.27E−01  8.41E−01 KEGG_TGF_BETA_SIGNALING_PATHWAY 85 0.3190 0.9616 5.47E−01  8.41E−01 KEGG_BETA_ALANINE_METABOLISM 22 0.4046 0.9468 5.33E−01  8.41E−01 KEGG_ERBB_SIGNALING_PATHWAY 86 0.3133 0.9462 5.80E−01  8.41E−01 KEGG_BLADDER_CANCER 40 0.3590 0.9449  5.46E−01 8.41E−01 KEGG_TRYPTOPHAN_METABOLISM 39 0.3580 0.9438  5.65E−01 8.41E−01 KEGG_GLYCOSYLPHOSPHATIDYLINOSITOL_GPI_ 25 0.3874 0.9353 5.43E−01  8.41E−01 ANCHOR_BIOSYNTHESISKEGG_GLYCINE_SERINE_AND_THREONINE_METABOLISM 30 0.3770 0.9349  5.68E−01 8.41E−01 KEGG_PENTOSE_AND_GLUCURONATE_INTERCONVERSIONS 23 0.3978 0.9310 5.80E−01  8.41E−01 KEGG_STEROID_BIOSYNTHESIS 16 0.4313 0.9285  5.80E−01 8.41E−01 KEGG_GLYCOSAMINOGLYCAN_DEGRADATION 21 0.3990 0.9236  5.81E−01 8.41E−01 KEGG_PATHWAYS_IN_CANCER 321 0.2739 0.9494  6.20E−01  8.70E−01KEGG_FC_EPSILON_RI_SIGNALING_PATHWAY 79 0.3108 0.9249  6.15E−01 8.70E−01 KEGG_CARDIAC_MUSCLE_CONTRACTION 73 0.3058 0.8963  6.30E−01 8.77E−01 KEGG_CYSTEINE_AND_METHIONINE_METABOLISM 33 0.3410 0.8707 6.63E−01  9.16E−01 KEGG_T_CELL_RECEPTOR_SIGNALING_PATHWAY 106 0.29150.8919  7.09E−01  9.26E−01 KEGG_GLIOMA 62 0.3116 0.8857  6.93E−01 9.26E−01 KEGG_ADHERENS_JUNCTION 68 0.3059 0.8855  6.97E−01  9.26E−01KEGG_FATTY_ACID_METABOLISM 42 0.3279 0.8719  6.83E−01  9.26E−01KEGG_BASAL_CELL_CARCINOMA 55 0.3100 0.8612  7.21E−01  9.26E−01KEGG_PENTOSE_PHOSPHATE_PATHWAY 26 0.3492 0.8408  7.11E−01  9.26E−01KEGG_BIOSYNTHESIS_OF_UNSATURATED_FATTY_ACIDS 20 0.3583 0.8338  7.01E−01 9.26E−01 KEGG_RIBOFLAVIN_METABOLISM 16 0.3816 0.8159  7.21E−01 9.26E−01 KEGG_ADIPOCYTOKINE_SIGNALING_PATHWAY 67 0.3025 0.8755 7.38E−01  9.41E−01 KEGG_GAP_JUNCTION 87 0.2863 0.8629  7.78E−01 9.58E−01 KEGG_TYPE_II_DIABETES_MELLITUS 46 0.3026 0.8226  7.91E−01 9.67E−01 KEGG_GLYCEROPHOSPHOLIPID_METABOLISM 71 0.2827 0.8316  8.21E−01 9.91E−01 KEGG_VASOPRESSIN_REGULATED_WATER_REABSORPTION 44 0.2918 0.8032 8.31E−01  9.96E−01 KEGG_TIGHT_JUNCTION 128 0.2691 0.8537  8.45E−01 1.00E+00 KEGG_GNRH_SIGNALING_PATHWAY 98 0.2748 0.8397  8.54E−01 1.00E+00 KEGG_MELANOGENESIS 98 0.2604 0.7985  9.00E−01  1.00E+00KEGG_THYROID_CANCER 29 0.3078 0.7626  8.66E−01  1.00E+00KEGG_DORSO_VENTRAL_AXIS_FORMATION 24 0.3171 0.7609  8.57E−01  1.00E+00KEGG_INSULIN_SIGNALING_PATHWAY 134 0.2351 0.7496  9.91E−01  1.00E+00KEGG_ENDOMETRIAL_CANCER 52 0.2653 0.7338  9.37E−01  1.00E+00KEGG_PEROXISOME 78 0.2441 0.7164  9.85E−01  1.00E+00KEGG_RENAL_CELL_CARCINOMA 66 0.2430 0.7008  9.72E−01  1.00E+00KEGG_NEUROTROPHIN_SIGNALING_PATHWAY 122 0.2183 0.6909  9.96E−01 1.00E+00 KEGG_B_CELL_RECEPTOR_SIGNALING_PATHWAY 74 0.2309 0.6826 9.98E−01  1.00E+00 KEGG_NOTCH_SIGNALING_PATHWAY 47 0.2500 0.6804 9.61E−01  1.00E+00 KEGG_AMINO_SUGAR_AND_NUCLEOTIDE_ 44 0.2466 0.6598 9.87E−01  1.00E+00 SUGAR_METABOLISM KEGG_VALINE_LEUCINE_AND_ISOLEUCINE_44 0.2416 0.6441  9.89E−01  1.00E+00 DEGRADATIONKEGG_MTOR_SIGNALING_PATHWAY 51 0.2221 0.6199  9.98E−01  1.00E+00KEGG_PROPANOATE_METABOLISM 32 0.2372 0.5923  9.89E−01  1.00E+00KEGG_HUNTINGTONS_DISEASE 170 0.1817 0.5895  1.00E+00  1.00E+00KEGG_PYRUVATE_METABOLISM 39 0.2174 0.5683  9.96E−01  1.00E+00KEGG_SYSTEMIC_LUPUS_ERYTHEMATOSUS 123 −0.7308 −2.3625 <1.00E−03<1.00E−02 KEGG_SPLICEOSOME 120 −0.6589 −2.1285 <1.00E−03 <1.00E−02KEGG_DNA_REPLICATION 36 −0.7853 −2.0793 <1.00E−03 <1.00E−02KEGG_CELL_CYCLE 124 −0.6132 −1.9829 <1.00E−03 <1.00E−02KEGG_HOMOLOGOUS_RECOMBINATION 26 −0.7502 −1.8667 <1.00E−03 <1.00E−02KEGG_RIBOSOME 87 −0.6122 −1.8588 <1.00E−03 <1.00E−02KEGG_RNA_DEGRADATION 56 −0.6176 −1.7888 <1.00E−03 <1.00E−02KEGG_OOCYTE_MEIOSIS 107 −0.5171 −1.6434 <1.00E−03 <1.00E−02KEGG_PROGESTERONE_MEDIATED_OOCYTE_MATURATION 85 −0.5121 −1.6010<1.00E−03 <1.00E−02 KEGG_BASE_EXCISION_REPAIR 33 −0.6575 −1.7074 2.19E−03  1.39E−02 KEGG_MISMATCH_REPAIR 23 −0.7278 −1.7707  2.36E−03 1.39E−02 KEGG_PROTEASOME 43 −0.5630 −1.5802  8.83E−03  5.32E−02KEGG_NUCLEOTIDE_EXCISION_REPAIR 44 −0.5541 −1.5345  1.53E−02  7.76E−02KEGG_PYRIMIDINE_METABOLISM 94 −0.4476 −1.3925  2.03E−02  8.83E−02KEGG_BASAL_TRANSCRIPTION_FACTORS 35 −0.5618 −1.4699  3.23E−02  1.21E−01KEGG_RNA_POLYMERASE 27 −0.5679 −1.4123  4.53E−02  1.51E−01KEGG_COLORECTAL_CANCER 62 −0.4119 −1.1964  1.75E−01  4.72E−01KEGG_PRIMARY_IMMUNODEFICIENCY 34 −0.4517 −1.1747  2.13E−01  5.24E−01KEGG_PRIMARY_BILE_ACID_BIOSYNTHESIS 16 −0.5303 −1.1852  2.27E−01 5.34E−01 KEGG_CITRATE_CYCLE_TCA_CYCLE 30 −0.4512 −1.1431  2.36E−01 5.48E−01 KEGG_SMALL_CELL_LUNG_CANCER 84 −0.3558 −1.1053  2.44E−01 5.59E−01 KEGG_PROXIMAL_TUBULE_BICARBONATE_RECLAMATION 23 −0.4753−1.1566  2.56E−01  5.74E−01 KEGG_PURINE_METABOLISM 152 −0.3191 −1.0691 2.68E−01  5.82E−01 KEGG_HISTIDINE_METABOLISM 27 −0.4347 −1.0768 3.10E−01  6.38E−01 KEGG_PHENYLALANINE_METABOLISM 18 −0.4584 −1.0618 3.65E−01  7.16E−01 KEGG_ALDOSTERONE_REGULATED_SODIUM_REABSORPTION 42−0.3858 −1.0507  3.77E−01  7.16E−01 KEGG_ETHER_LIPID_METABOLISM 30−0.4050 −1.0352  4.00E−01  7.46E−01KEGG_GLYOXYLATE_AND_DICARBOXYLATE_METABOLISM 16 −0.4608 −1.0148 4.14E−01  7.63E−01 KEGG_PROSTATE_CANCER 89 −0.3204 −0.9982  4.27E−01 7.63E−01 KEGG_HEDGEHOG_SIGNALING_PATHWAY 56 −0.3540 −1.0074  4.30E−01 7.63E−01 KEGG_P53_SIGNALING_PATHWAY 66 −0.3441 −1.0082  4.49E−01 7.83E−01 KEGG_ONE_CARBON_POOL_BY_FOLATE 17 −0.4277 −0.9850  4.88E−01 8.33E−01 KEGG_GLUTATHIONE_METABOLISM 48 −0.3354 −0.9456  5.51E−01 8.40E−01 KEGG_TYROSINE_METABOLISM 41 −0.3461 −0.9432  5.59E−01 8.40E−01 KEGG_TERPENOID_BACKBONE_BIOSYNTHESIS 15 −0.4210 −0.9251 5.66E−01  8.40E−01 KEGG_NON_SMALL_CELL_LUNG_CANCER 54 −0.3267 −0.9320 5.75E−01  8.40E−01 KEGG_SELENOAMINO_ACID_METABOLISM 24 −0.3674 −0.9087 5.94E−01  8.52E−01 KEGG_VIBRIO_CHOLERAE_INFECTION 53 −0.3177 −0.9225 5.98E−01  8.52E−01 KEGG_ALPHA_LINOLENIC_ACID_METABOLISM 19 −0.3620−0.8293  7.19E−01  9.24E−01 KEGG_LONG_TERM_DEPRESSION 70 −0.2922 −0.8691 7.45E−01  9.43E−01 KEGG_ACUTE_MYELOID_LEUKEMIA 57 −0.2957 −0.8524 7.58E−01  9.53E−01 KEGG_CHRONIC_MYELOID_LEUKEMIA 73 −0.2905 −0.8710 7.70E−01  9.60E−01 KEGG_PANCREATIC_CANCER 69 −0.2928 −0.8606  7.77E−01 9.60E−01 KEGG_AMYOTROPHIC_LATERAL_SCLEROSIS_ALS 52 −0.2946 −0.8457 8.00E−01  9.72E−01 KEGG_O_GLYCAN_BIOSYNTHEIS 27 −0.2958 −0.7509 8.78E−01  1.00E+00 KEGG_UBIQUITIN_MEDIATED_PROTEOLYSIS 131 −0.2490−0.8209  9.31E−01  1.00E+00 KEGG_ARGININE_AND_PROLINE_METABOLISM 48−0.2670 −0.7389  9.36E−01  1.00E+00 KEGG_BUTANOATE_METABOLISM 34 −0.2643−0.6990  9.55E−01  1.00E+00 KEGG_PROTEIN_EXPORT 22 −0.2643 −0.6293 9.74E−01  1.00E+00 KEGG_PARKINSONS_DISEASE 112 −0.2391 −0.7645 9.74E−01  1.00E+00 KEGG_VEGF_SIGNALING_PATHWAY 75 −0.2379 −0.7151 9.77E−01  1.00E+00 KEGG_LYSINE_DEGRADATION 39 −0.2282 −0.6218  9.89E−01 1.00E+00 KEGG_AMINOACYL_TRNA_BIOSYNTHESIS 41 −0.2402 −0.6572  9.90E−01 1.00E+00 KEGG_GLYCOLYSIS_GLUCONEOGENESIS 61 −0.2270 −0.6631  9.93E−01 1.00E+00 KEGG_PORPHYRIN_AND_CHLOROPHYLL_METABOLISM 37 −0.2269 −0.6034 9.96E−01  1.00E+00 KEGG_OXIDATIVE_PHOSPHORYLATION 116 −0.1799 −0.5790 1.00E+00  1.00E+00

TABLE 6 Summary of Qiagen PCR array analysis SEN (L) cells 3X cellsNumber Number of genes Percent¹ of genes Percent¹ Total genes 84 100% 84 100%  Upregulated genes 77 92% 80 95% Downregulated genes 7  8% 4  5%Changing genes passing filters² 58 69% 44 52% Passing genes upregulated57 68% 44 52% Passing genes downregulated 1  1% 0  0% Changing genesfailing filters 26 31% 40 48% Genes passing filters, unique³ 23 27% 911% Genes passing filters, overlap⁴ 35 42% 35 42% Genes passing filters,total⁵ 67 80% 67 80% ¹All percentages are calculated with respect to thetotal number of genes (84) found on the array. Data for all 84 genesdisplayed as scatter plots are shown in FIG. 2h. ²The sum of upregulatedand downregulated genes that pass a set of significance filters, seeMethods for definitions of filters. ³Changing genes that pass thesignificance filters that are unique to either SEN (L) or 3X cells.⁴Changing genes that pass the significance filters that are common to(found in both) SEN (L) and 3X cells. ⁵Changing genes that pass thesignificance filters that are found in SEN (L) and/or 3X cells. Theheatmap representation for this set of genes (67) is shown in ExtendedFIG. 4j, k.

TABLE 7 Age-group Foldchanges L1 p16 IFNA IRF7 OAS1 IL6 MMP3 PAI 05mo-WT 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 05 mo-3TC 1.4200.868 1.489 0.922 2.229 2.046 0.993 1.265 26 mo-WT 6.265 2.917 13.2191.560 1.898 8.775 3.078 6.603 26 mo-3TC 3.947 2.213 6.579 1.076 1.6812.355 1.762 2.839 T-TEST 05 VS 3TC 0.152 0.819 0.304 0.542 0.125 0.1230.981 0.547 05 VS 26 0.001 0.001 0.000 0.001 0.207 0.001 0.022 0.110 26VS 3TC 0.068 0.129 0.016 0.070 0.786 0.001 0.090 0.194 IndividualFoldchanges L1 p16 IFNA IRF7 OAS1 IL6 MMP3 PAI 05 mo-WT 1.013 0.3720.874 1.062 0.684 0.953 0.488 0.645 1.249 0.342 0.982 0.868 0.769 1.0591.011 0.916 0.884 0.412 0.394 0.835 1.441 1.003 0.663 0.695 1.343 3.4191.540 0.953 0.791 0.848 1.660 1.291 1.085 1.107 1.037 1.079 0.355 0.8810.755 2.511 0.755 0.552 1.886 1.397 1.799 1.352 1.971 0.314 0.979 1.4080.557 0.679 0.977 1.112 0.913 0.596 0.692 0.388 0.729 1.127 1.183 0.7920.539 1.033 05 mo-3TC 1.458 2.881 1.821 1.040 2.867 1.455 1.587 2.2911.464 0.457 2.748 1.153 1.521 0.924 0.601 0.842 0.484 0.957 2.909 1.1326.031 5.604 1.946 2.212 1.829 0.270 0.645 0.684 1.317 1.622 0.711 1.6832.537 0.104 0.465 0.565 0.037 1.920 0.635 0.180 0.747 0.541 0.346 0.9601.603 0.751 0.476 0.380 26 mo-WT 10.645 2.563 17.947 1.801 5.442 17.0586.449 0.979 3.930 1.799 1.336 1.478 0.423 3.264 1.275 1.842 1.688 4.4418.010 1.439 0.531 3.254 2.541 3.243 10.959 2.315 22.657 1.891 0.51619.464 5.831 32.301 13.251 4.668 24.009 1.007 4.476 10.752 6.948 5.1592.413 4.457 17.239 2.233 0.231 4.028 1.564 0.306 4.278 1.361 12.8791.461 1.699 4.075 0.764 9.405 2.796 2.689 0.531 1.971 4.666 7.083 1.16116.692 6.245 3.037 13.076 1.660 2.248 8.622 3.317 1.188 4.788 1.77710.981 1.422 0.963 3.754 1.551 1.605 6.397 2.575 13.747 1.240 0.59814.603 0.982 4.307 7.793 3.327 16.220 1.120 0.986 9.347 4.554 2.206 26mo-3TC 2.819 2.290 10.115 0.540 1.658 1.116 1.044 1.034 2.160 3.6532.941 0.506 0.643 1.553 0.972 1.056 2.053 1.731 4.156 0.498 0.055 2.1941.618 2.696 1.491 1.168 1.283 0.424 0.482 0.686 1.650 0.493 6.524 0.8441.601 0.954 1.094 1.395 1.033 1.562 2.863 1.243 8.335 0.831 0.695 1.2911.166 1.675 7.191 3.690 19.836 0.891 7.091 8.067 4.484 2.347 5.433 2.4583.982 3.302 3.841 0.654 0.374 0.529 3.817 2.135 6.531 0.993 1.945 2.1191.543 0.747 4.409 4.053 8.332 1.155 0.636 3.177 3.656 8.508 5.864 1.9695.514 0.950 0.565 1.148 1.964 6.112 2.745 1.326 6.321 1.866 1.469 4.8661.637 7.313

TABLE 8 NRTIs Approved as Anti-HIV Therapies Trade Name Generic NameFormulation Std Adult Dose Nucleoside/nucleotide reverse transcriptaseinhibitors (NRTIs) ZIAGEN ™ Abacavir 300 mg tablet 300 mg twice a day or600 mg once a day EMTRIVA ™ Emtricitabine 200 mg capsule 200 mg once aday EPIVIlR ™ Lamivudine 150 and 300 mg tablets 150 mg twice a day or300 mg once a day RETROVIR ™ Zidovudine 100 and 250 mg capsules 100 mg 5times a day or 250 mg twice a day VIREAD ™ Tenofovir disoproxil 245 mgtablet 245 mg once a day NRTI fixed-dose combinations KIVEXA ™Abacavir/lamivudine Tablet comprising: 600 mg abacavir and One tabletonce a day 300 mg lamivudine TRIZIVIR ™ Abacavir/lamivudine/ Tabletcomprising: 300 mg abacavir, One tablet once a day zidovudine 150 mglamivudine and 300 mg zidovudine TRUVADA ® Emtricitabine/tenofovirTablet comprising: 200 mg emtricitabine One tablet once a day disoproxiland 245 mg tenofovir disoproxil DESCOVY ® Emtricitabine/tenofovir Tabletcomprising: 200 mg emtricitabine One tablet once a day alafenamide and10 or 25 mg tenofovir alafenamide COMBIVIR ™ Lamivudine/zidovudineTablet comprising: 150 mg lamivudine and One tablet once a day 300 mgzidovudine ATRIPLA ® Efavirenz/emtricitabine/ Tablet comprising: 600 mgefavirenz, One tablet once a day tenofovir disoproxil 200 mgemtricitabine, and 245 mg tenofovir disoproxil EVIPLERA ™Rilpivirine/emtricitabine/ Tablet comprising: 25 mg rilpivirine, Onetablet once a day tenofovir disoproxil 200 mg emtricitabine, and 245 mgtenofovir disoproxil ODEFSEY ® Rilpivirine/tenofovir Tablet comprising:25 mg rilpivirine, One tablet once a day alafenamide/emtricitabine 25 mgtenofovir alafenamide, and 200 mg emtricitabine GENVOYA ®Elvitegravir/cobicistat/ Tablet comprising: 150 mg elvitegravir, Onetablet once a day emtricitabine/tenofovir 150 mg cobicistat, 200 mgemtricitabine, alafenamide 10 mg tenofovir alafenamide STRIBILD ®Elvitegravir/cobicistat/ Tablet comprising: 150 mg elvitegravir,emtricitabine/tenofovir 150 mg cobicistat, 200 mg emtricitabine, Onetablet once a day disoproxil and 245 mg tenofovir disoproxil TRIUMEQ ®Dolutegravir/abacavir/ Tablet comprising: 50 mg dolutegravir, One tabletonce a day lamivudine 600 mg abacavir, and 300 mg lamivudine

TABLE 9 Lower dosage (50%) of NRTIs Trade Name Generic Name FormulationStd Adult Dose Nucleoside/nucleotide reverse transcriptase inhibitors(NRTIs) ZIAGEN ™ Abacavir 150 mg tablet 150 mg twice a day or 300 mgonce a day EMTRIVA ™ Emtricitabine 100 mg capsule 100 mg once a dayEPIVIR ™ Lamivudine 75 and 150 mg tablets 75 mg twice a day or 150 mgonce a day RETROVIR ™ Zidovudine 50 and 125 mg capsules 50 mg 5 times aday or 125 mg twice a day VIREAD ™ Tenofovir disoproxil 120 mg tablet120 mg once a day NRTI fixed-dose combinations KIVEXA ™Abacavir/lamivudine Tablet comprising: 300 mg abacavir and One tabletonce a day 150 mg lamivudine TRIZIVIR ™ Abacavir/lamivudine/ Tabletcomprising: 150 mg abacavir, One tablet once a day zidovudine 75 mglamivudine and 150 mg zidovudine TRUVADA ® Emtricitabine/tenofovirTablet comprising: 100 mg emtricitabine One tablet once a day disoproxiland 120 mg tenofovir disoproxil DESCOVY ® Emtricitabine/tenofovir Tabletcomprising: 100 mg emtricitabine One tablet once a day alafenamide and 5or 12 mg tenofovir alafenamide COMBIVIR ™ Lamivudine/zidovudine Tabletcomprising: 75 mg lamivudine and One tablet once a day 150 mg zidovudineATRIPLA ® Efavirenz/emtricitabine/ Tablet comprising: 300 mg efavirenz,One tablet once a day tenofovir disoproxil 100 mg emtricitabine, and 120mg tenofovir disoproxil EVIPLERA ™ Rilpivirine/emtricitabine/ Tabletcomprising: 12 mg rilpivirine, One tablet once a day tenofovirdisoproxil 100 mg emtricitabine, and 120 mg tenofovir disoproxilODEFSEY ® Rilpivirine/tenofovir Tablet comprising: 12 mg rilpivirine,One tablet once a day alafenamide/emtricitabine 12 mg tenofoviralafenamide, and 100 mg emtricitabine GENVOYA ® Elvitegravir/cobicistat/Tablet comprising: 75 mg elvitegravir, One tablet once a dayemtricitabine/tenofovir 75 mg cobicistat, 100 mg emtricitabine,alafenamide 10 mg tenofovir alafenamide STRIBILD ®Elvitegravir/cobicistat/ Tablet comprising: 75 mg elvitegravir, Onetablet once a day emtricitabine/tenofovir 75 mg cobicistat, 100 mgemtricitabine, and disoproxil 120 mg tenofovir disoproxil TRIUMEQ ®Dolutegravir/abacavir/ Tablet comprising: 25 mg dolutegravir, One tabletonce a day lamivudine 300 mg abacavir, and 150 mg lamivudine

TABLE 10 Mouse L1 activity inhibition IC₅₀ (μM), n = 2 Compounds Exp. 1Exp. 2 Average Lamivudine 0.958 0.780 0.869 Stavudine 1.590 1.856 1.723Empricitabine 0.640 0.451 0.546 Apricitabine 4.442 3.825 4.134 TenofovirDisiproxil 0.157 0.151 0.154 Tenofovir 3.467 4.024 3.746 Censavudine0.118 0.105 0.112 Elvucitabine 0.116 0.106 0.111 IC₅₀ determination fornine compounds to inhibit the retrotransposition activity of activemouse LINE-1 in HeLa cells. Retrotransposition activity was determinedwith the dual luciferase pYX016 reporter. Cells were treated withdifferent concentrations of each compounds and transfected with pYX016at the same time. Luminescence was measured at 48 H post- transfection.The experiment was performed two times independently.

TABLE 11 Human L1 activity inhibition IC₅₀ (μM), n = 2 Compounds Exp. 1Exp. 2 Average Lamivudine 0.793 0.867 0.830 Censavudine 0.053 0.0920.072 Elvucitabine 0.094 n/a n/a IC₅₀ determination for three compoundsto inhibit the retrotransposition activity of active human LINE-1 inHeLa cells. Retrotransposition activity was determined with the dualluciferase pYX017 reporter. Cells were treated with differentconcentrations of each compounds and transfected with pYX017 at the sametime. Luminescence was measured at 72 H post- transfection. Theexperiment was performed two times independently.

TABLE 12 Human L1 activity inhibition Human LINE-1 IC50 (μM), n = 3Compounds Exp.1 Exp.2 Exp. 3 Average Lamivudine 0.90 0.70 N/A 0.80Censavudine 0.13 0.12 0.05 0.10 Bictegravir* 12.41 >50 N/A >12.41Efavirenz >50 >0.25 N/A >0.25 Nevirapine >50 >50 N/A >50Rilpivirine* >50 15.08 45.49 >15.08 Zidovudine 0.41 0.34 1.68 0.81Islatravir N/A 1.18E−03 1.40E−03 1.29E−03 Raltegravir potassium 9.86 >5049.28 >9.86 Dolutegravir sodium 9.38 N/A >20 >9.38

TABLE 13 Comparative Studies Tenofovir Lamivudine EmtricitabineTenofovir disoproxil Stavudine Abacavir (3TC) (FTC) (TFV) (TDF) (d4T)Islatravir (ABC) Human IC₅₀ (uM) 0.64 1.34 2.77 0.20 0.75 0.005 17.1Line-1 Human Intracellular 9% 67% 0.66% N/A 63% TBD 106% Line-1 drugconc. (% of IC-50 conc.) huCSF/Plasma 0.42 0.35 0.02 N/A 0.2 0.25 0.67

TABLE 14 Human LINE-1 Inhibitory Quotient (IQ) Human LINE-1 InhibitoryQuotient (IQ) PBMC Brain (EFdA-TP EC50 = 316 nM) (assume Br./plasma =0.25) single dose q.d. single dose q.d. Dose (mg) IQ C_(168 hr) IQC_(avg (ss)) IQ C_(168 hr) IQ C_(avg (ss)) 0.5 1.84 23.41 0.46 5.85 12.61 39.79 0.66 9.95 2 3.00 50.54 0.75 12.64 10 15.64 295.15 3.92 73.7930 76.88 915.31 19.22 228.83

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All patents, patent application, and publications cited herein are fullyincorporated by reference herein.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections may set forth one or morebut not all exemplary embodiments of the present invention ascontemplated by the inventor(s), and thus, are not intended to limit thepresent invention and the appended claims in any way.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments but should be definedonly in accordance with the following claims and their equivalents.

What is claimed is:
 1. A method for treating a disease, condition, ordisorder in a patient in need thereof, the method comprisingadministering a therapeutically effective amount of islatravir,censavudine, or elvucitabine to the patient, wherein the islatravir,censavudine, or elvucitabine prevents or reverses at least one of theupregulation of LINE-1 (L1), the accumulation of cytoplasmic L1 cDNA,the activation of an type-I interferon (IFN-I) response, or thesenescence associated secretory phenotype (SASP) pro-inflammatory statein the patient, and the disease, condition, or disorder is Alzheimer'sdisease, amyotrophic lateral sclerosis (ALS), atherosclerosis, autismspectrum disorder (ADS), autoimmune disease, cardiovascular dysfunction,dementia with lewy Bodies (DLB), frontotemporal dementia (FTD), hearingloss, hematopoietic stem cell function, Huntington's disease, mildcognitive impairment (MCI), multi systems atrophy (MSA), corticobasaldegeneration (CDB), multiple sclerosis (MS), osteoarthritis,osteoporosis, Parkinson's disease, peripheral degenerative disease,progressive supra nuclear palsy (PSP), pulmonary fibrosis, RettSyndrome, schizophrenia, vision loss, or Aicardi-Goutieres syndrome(AGS).
 2. The method of claim 1, wherein the disease, condition, ordisorder is Alzheimer's disease, ALS, FTD, PSP, or Rett Syndrome.
 3. Themethod of claim 2, wherein the disease, condition, or disorder isAlzheimer's disease or ALS, and the patient experiences a decrease inone or more symptoms of Alzheimer's disease or ALS after administrationof islatravir, censavudine, or elvucitabine to the patient.
 4. Themethod of claim 3, wherein the disease, condition, or disorder isAlzheimer's disease, and the one or more symptoms comprise memory loss,misplacing items, forgetting the names of places or objects, repeatingquestions, being less flexible, confusion, disorientation, obsessivebehavior, compulsive behavior, delusions, aphasia, disturbed sleep, moodswings, depression, anxiety, frustration, agitation, difficulty inperforming spatial tasks, agnosia, difficulty with ambulation, weightloss, loss of speech, loss of short term memory or loss of long termmemory.
 5. The method of claim 3, wherein the disease, condition, ordisorder is Alzheimer's disease, and the decrease of symptoms isdetermined using the cognitive subscale of the Alzheimer's diseaseAssessment Scale (ADAS-cog), the Clinician's Interview-Based Impressionof Change (CIBIC-plus), or the Activities of Daily Living Scale (ADL).6. The method of claim 1, wherein the underlying pathology of thedisease, condition, or disorder is identified by detection of abiomarker before and after administration of islatravir, censavudine, orelvucitabine to the patient.
 7. The method of claim 6, wherein thebiomarker is β-amyloid or Tau protein.
 8. The method of claim 1, whereinthe disease, condition, or disorder is an autoimmune disease selectedfrom Aicardi Goutiere's Syndrome (AGS), lupus, or rheumatoid arthritis.9. The method of claim 1, further comprising administering atherapeutically effective amount of at least one second therapeuticagent useful for treating the disease, condition, or disorder.
 10. Themethod of claim 9, wherein the at least one second therapeutic agent isabacavir, a combination of abacavir and lamivudine, a combination ofabacavir, lamivudine, and zidovudine, a combination of lamivudine andzidovudine, lamivudine, zidovudine, a combination of emtricitabine andtenofovir disoproxil fumarate, emtricitabine, tenofovir disoproxilfumarate, a combination of emtricitabine and tenofovir alafenamide,didanosine, stavudine, apricitabine, alovudine, dexelvucitabine,amdoxovir, fosalvudine, elvucitabine, efavirenz, nevirapine,delavirdine, etravirine or rilpivirine.
 11. The method of claim 9,wherein the disease, condition, or disorder is Alzheimer's disease ormild cognitive impairment, and the at least one second therapeutic agentis donepezil, galantamine, rivastigmine, memantine, bapineuzumab,ABBV-8E12, CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293),LY2886721, nicotinamide, or MPT0G211.
 12. The method of claim 9, whereinthe disease, condition, or disorder is ALS, and the at least one secondtherapeutic agent is edaravone, riluzole, raltegravir, curcumin,derivatives of curcumin, chicoric acid, derivatives of chicoric acid,3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic acid,aurintricarboxylic acid, derivatives of aurintricarboxylic acid, caffeicacid phenethyl ester, derivatives of caffeic acid phenethyl ester,tyrphostin, derivatives of tyrphostin, quercetin, derivatives ofquercetin, S-1360, zintevir (AR-177), L-870812, L-25 870810, MK-0518,BMS-538158, or GSK364735C.
 13. The method of claim 1, wherein thepatient is (a) not infected with the HIV virus, (b) is not suspected ofbeing infected with the HIV virus, and/or (c) is not being treated toprevent infection with the HIV virus.
 14. The method of claim 1comprising administering a therapeutically effective amount ofislatravir to the patient in need thereof.
 15. The method of claim 14,wherein islatravir is administered daily in an amount of about 0.1 mg toabout 10 mg.
 16. The method of claim 1 comprising administering atherapeutically effective amount of censavudine to the patient in needthereof.
 17. The method of claim 16, wherein the disease, condition, ordisorder is PSP, ALS, or AGS.
 18. The method of claim 1 comprisingadministering a therapeutically effective amount of elvucitabine to thepatient in need thereof.
 19. A method for treating PSP, ALS, or AGS in apatient in need thereof, the method comprising administering censavudineto the patient in amount sufficient to prevent or reverse at least oneof the upregulation L1, the accumulation of cytoplasmic L1 cDNA, theactivation of an IFN-I response, or the SASP pro-inflammatory state inthe patient.
 20. A method for treating PSP, ALS, or AGS in a patient inneed thereof, the method comprising administering islatravir orelvucitabine to the patient in amount sufficient to prevent or reverseat least one of the upregulation L1, the accumulation of cytoplasmic L1cDNA, the activation of an IFN-I response, or the SASP pro-inflammatorystate in the patient.
 21. The method of claim 16, wherein the disease,condition, or disorder is Alzheimer's disease, ADS, FTD, MS, Parkinson'sdisease, Rett Syndrome, or schizophrenia.
 22. The method of claim 21,wherein the disease, condition, or disorder is FTD.
 23. The method ofclaim 21, wherein the disease, condition, or disorder is Alzheimer'sdisease.
 24. The method of claim 17, wherein the disease, condition, ordisorder is PSP.
 25. The method of claim 17, wherein the disease,condition, or disorder is ALS.
 26. The method of claim 17, wherein thedisease, condition, or disorder is AGS.